6

Mucopolysaccharides: Chemistry.

Biosynthesis and Metabolism

Albert Dorfman

1. Physiology and Composition of Conncctivc Tissuc

It is a great honor for me to have the opportunity to visit Japan and to be able to address the numerous Japanese physicians and scientists who have contributed so much to our knowledge of medicine and biochemistry.. It is， however， a particular saddness to me that my friend， Professor Hajime Masamune， cannot be present with us on this occasion.

Professor Masamune's contributions to medicine will be eternally remembered. Over many years he labored hard and effectively to increase our knowledge of the carbohydrate components of tissues. During this time he was frequently in the fore-front of discovery of new knowledge. In some cases it was many years before the importance of his discoveries was appreciated.

His demonstration of the relationships of the blood group substances has now become recognized throughout the world as an important pioneer effort. In the field of the acid mucopolysaccharides he pioneered in the discovery of the true structure of chondroitinsulfuric acid. Had his earlier pap巴rsbeen better appreciated much work and time might have been saved. He was the first to point out that there existed a chondroitinsulfuric acid which had a uronic acid different from glucuronic acid. It was only many years later that others， including our own laboratory， appreciated the significance of this great discovery. Throughout his career， these individual contributions formed a pattern of a devoted love of scientific investigation which is typical of all great scientists. The contributions that 1 mentioned are only a few of his many contributions to mankind. It is， therefore， a great honor to me to come to meet with his many students and see his important influence in his own country.

1 consider it a great good fortune that Professor Masamune visited our labora-tories in Chicago. We were all struck， not only by his scientific wisdom， but by his human kindness. It is a great saddness that he cannot be present with us for this series of lectures which were really his conception. However， 1 am deeply honored to be able to pay tribute to him by dedicating these lectures to his memory.

In the course of these lectures it is my hope to present certain concepts regarding that compartment of various animal species which is usually known as connective tissue. The recent progress in the understanding of enzymatic mechanisms involved in biosynthesis and energy transfer has focussed attention on the intercellular reac-tions. This has been particularly true in the past several years.

During this time we have witnessed the startling discovery of enzymatic mecha-nisms involved in polysaccharide， protein， nucleic acid and lipid synthesis and we have learned the detailed mechanisms by which energy is made available to the cell

7

for its many important functions. Already the entire field of nutrition has become a part of enzyme chemistry so that we have a real understanding of the function of trace compounds in mammalian metabolism. There are indications that we shall soon know much more about the effect of endocrine agents on the metabolic cycles in tissues.

In the course of these start1ing discoveries less attention has been paid to the milieu in which the cell metabolizes and the factors which control.the composition both qualitative and quantitative of this milieu. We now know that changes in the environment of the cell result in profound changes within the cell and that this medium surrounding cells everywhere is of critical importance in health and dis-ease. The increasing attention to the diseases， commonly known as connective tissue diseases， or collagen diseases， has resulted in reawakening of interest in the connective tissues of the body， for it is in these tissues that the pathologist most strikingly observes the changes in these diseases.

Of even greater bioloigcal and medical significance is the fact that it is the degeneration of connective tissue and its vascular component which typifies the ageing process. It is perhaps in these areas that one must look to determine the essential nature of ageing.

An approach to an understanding of the function of connective tissue involves not only the well-developed structure in the complex mammalian organism， but a consideration of the physiology of cells in general and their evolution from the simplest forms. The cytoplasm of unicellular organisms is separated from its sur-rounding medium by a simple cellular membrane. The nature of this cellular mem-brane varies widely in different types of unicellular organisms. The entire com-position of cell membranes is as yet unknown， but the presence of a complex lipid and protein structure and properties of semi-permeability are established. In certain types of cells this protection of the cytoplasm is morecomplex. Witness the fact that in bacteria a complex cell wall has been developed which is actually an addi tion to the cell membrane. It is of considerable interest that the cell wall of mi-croorganisms is a highly specialized structure both anatomically and chemically and that it contains certain of the constituents that are found in the polymers char-acteristic of connective tissue of higher organisms. 1 refer， of c

8

Fig. 1. A schematic l'epresentation of an idealized connective tissue.

for protection and support. 1n the simplest colonial forms and two germ layer or-ganisms， a mucinous-like material appears to serve this function. Even in simpler forms mucinous material in itself apparently does not afford the necessary structural stability and fibers appear. These fibers show some of the characteristics that are to be found in the more complex animals and are identifiable as collagen. Although these fibers do not have a chemical composition of collagen of the mammal， the physical structure， as indicated by X-ray diffraction and electron microscopy appear-ance. is similar to mammalian collagen1l.

With increasing complexity. the system by which homeostasis must be maintained becomes more and more complex so that finally we have the development not only of more complex connective tissue with its specialized mesodermal cells but a circulatory system which affords a channel between the exterior of the body and the connective tissuc. At the same time connective tissue. with its functions of support and homeostasis， becomes more and ffiore specialized so that a variety of different connective tissues evolves-each with its own specialized structure. Witness the contrast between the structure and composition of the synovial fiuid which has become highly specialized as a means of lubrication， and bone to which has been added calcium to afford rigid support.

This general introduction has been meant to place in some perspective the chem-ical and physical evolution of the connective tissues of the body and to point out their role in the control of metabolism of organisms of all types. It is particularly interesting to appreciate the diversity and yet chcmical unity which nature has a-chieved for the protection of the cell.

Our further consideration will be more restricted for we will be concerned more specifically with certain aspects of the connective tissues of the mammalian organisms

9

which have been studied most thoroughly. Figure 1 illustrates an idealized version of the structure of connective tissue. This is not a photomicrograph， but rather represents an idealized version of what the chemist would prefer to consider as connective tissue. The cells to the left are meant to illustrate parenchymal cel1s of any kind. These cells which perform干italmetabolic functions are immersed in connective tissue. To the right is shown a capillary-a portion of the connec-tive tissue in one respect and of the circulation in another， whic.h is bringing to the cell nutrients from the exterior and taking from the cell the waste products of metabolism. Separating the capillary and parenchymal cells lies the connective tissue barrier.

Two cell typ巴speculiar to connective tissue are d巴monstrated. This does not mean to indicate that other cells in connective tissue do not play vital functions. It may be that in the process of histogen巴sisa common cell type leads to the devel-opment of specialized mesodermal cells each with its own function. The two types of cells with which we wi11 be concerned are the mast cell and the fibroblast. The older studies， starting with the now c1assical work of Maximowv i11ustrate that the fibroblast， when grown in cu1ture， is the source of production of collagen. More recent1y a number of studies have indicated that these cells are able to produce a diversity of polysaccharides when grown in tissue culture. These inc1ude at least several of the known acid mucopolysaccharides3山町 Itseems likely that other spe-cialized types of fibroblasts wi11 be shown to produce the remainder of the known acid mucopolysaccharides. Many important questions remain to be considered re-garding this process. Does the most primitive cell have the potentia1ity of producing all of the polysaccharides? How do they differentiate so that they produce specific polysaccharides in specific localizations? Is this a process by which c巴rtainenzy-matic steps are lost so that only a limited number of polysaccharides are avai1able，

or do these cells still r巴tainthis capacity and is production stimulated by local fac-tors? These and many other important questions of chemical differentiation remain for future investigation.

A second type of cell which has received great attention is the mast cell. This cell， the fixed tissue basophil， characterized by large basophilic-staining granules. Ther

10

secretory function of the mast cell. Characteristic also of connective tissues in most locations are the collagen fibers.

The structure of collagen fibers has occupied the altention of many investigators. During recent years great progress has been made， largely due to the improved physical chemical methods which are now availablcωThus， the electron micro-scope picture has indicated specific characteristics of the collagen fi.ber as illustrated in Figure 2. Morc recent studies have shown even greater resolution of this char-

Fig. 2. Electron micrograph of cOIlagen fibers teased from guinea pig AcbiIles tendon in water. (Reproduced from : X-ray and Electron Microscope Studies of the Structure of COllagen Fibers， Schmitt， F'rancis 0.， ]. Am. Leather Chemists Assoc.， XXXIX. 430 (1944)).

Fig. 3. An illustration of the possible al'l'angement of the 3 chains of collagen in helix. The figure to the left is a structure pro-posed fol' colIagen 1 and that in the middle for collagen II. The ugure to the l'ight shows the turn of the right hand helices around a common axis indicated by a dotted line.

n

acteristic banded appearance of col1agen. Considerable information regarding the structure of col1agen has been added in recent years by the studies of soluble colra-gens which have now been prepared from a variety of sources.

It is apparent that reticulin has a chemical composition similar to col1agen. The factors are unknown which determine the formation of smal1 reticular fibers in certain locations in contrast to the large col1agen bundles in others.

The molecular structure of col1agen is still imperfectly understood but the pres-ent concept indicates that it consists of 3 helices-each representing a peptide chain which are fitted together to form the basic col1agen fibril. The manner in which these helices are arranged still is uncertain. A proposal of this arrangement has been made by Rich and Crick10

) and is shown in Figure 3. Chemical and biological understanding of col1agen has received considerable impetus from the recognition of soluble forms of col1agen. The demonstration of acid soluble col1agen. original1y called procollagen. revived interest in the finding by NageotteJ1l of the existence of acetic acid soluble col1agen 12 - W • More recent studies have focussed attention on neutral salt soluble col1agen. Numerous investigations have established that soluble forms of col1agen are converted to typical col1agen fibers by variations in salt con-centration. pH. temperature and other additives15-18). Under certain conditions collagen may be transformed to structures different from those present in natural materials. The exact nature of an individual collagen molecule is not clear. nor is the nature of the transition of soluble collagen to the collagen fibril. Doty and coworkers19) have studied the molecular parameters of soluble collagen. This “tropo・collagen" molecule is highly asymmetric with a molecular weight of 350.000. a length of 3.000.A. and a diameter of 13.5.A. These dimensions are approximately the same for ichythycoI. calf skin. and cod skin soluble collagens. Similar measurements have been made for rat skin collag巴nand rat tendon soluble col1agen.

Knowledge concerning both the chemistry and biology of elastin fibers is limited. Elastin has amino acid composition strikingly diff巴rentfrom collagen and charac-terized by a low content of polar amino acids20，211. No characteristic spacing is ob-served in the electron microscope and no evidence of orientation

12

The remainder of these lectures will be concerned with a more detailed discus旬

sion of some of the properties of the acid mucopolysaccharides-a group of substances found in the connective tissues of the body. These materials are high polymers composed of alternating units of a uronic acid and an N-acetylhexosamine. It seems likely that in the body， long chains of alternating polysaccharide units exist. In some cases it is apparent that these are linked to a specific protein which differs from collagen in its structure. The basic unit of an acid mucopolysaccharide is

H

H

H OH

O o"， ll/oH 、~V

c片側O

H

Q

NHAc

Fig. 4. Structure of chondroitinsulfuric acid-A.

H，OH

illustrated in the next figure (Figure 4) showing the repeating disaccharide unit of chondroitinsulfuric acid押A. Chondroitinsulfuric acid-A ls the chondroitin sulfate of most mammalian cartilage and has been known longer than any of the other acid mucopolysaccharides. Its exact structure was a matter of concern for many years since the early pioneering work of Levene and his co-workers. The structure as we know it now was established by Davidson and Meyer2Sl， and to a considerable extent depended upon the earlier findings of Professor Masamune and his colleagues. It should be noted that this structure represents a disaccharide consisting of a1ter-nating units of glucuronic acid and N-acetylgalactosamine. Glucuronic acid is linked from the first carbon atom to the third carbon atom of the N-acetylgalactosamine，

while the first carbon atom of N-acetylgalactosamine is linked tci the 4-position of the uronic acid. This type of linkage appears to be present in hyaluronic acid and is probably present in chondroitinsulfuric acids-B and -C， as well. Of special note is the occurrence of an ester sulfate group in the 4-position. The presence of an ester sulfate group and a carboxyl group results in the presence of negative charge at each of the monosaccharide units， a property which produces the distinctive char-acteristics of the acid mucopolysaccharides.

In earlier conceptions of connective tissue ground substance， many investigators considered these substances as a general c1ass with similar properties so that much of the biological literature still is concerned with the general properties of ground substance as if it were a specific chemical substance. It is now known that the acid mucopolysaccharides consist of a diverse group of compounds with similar and yet di妊erentstructures.

In Table 1 is listed the known acid mucopolysaccharides together with their component sugars. The first on this list of polysaccharides is hyaluronic acid-a polysaccharide that has been found in a wide variety of tissues. This polysaccha-ride is composed of alternating units of N-acetylglucosamine and glucuronic acid and does not have a sulfate group. It is found in abundance in synovial fluid， vitreous humor， and is also present， mixed with other polysaccharides， in skin and other tissues. The linkage of this substance to protein is still somewhat uncertain. This

13

Mucopolysaccharides of Connective Tissue Table 1

Antithrombic Activity

O

O

+ O q

O(?)

+ 守

Sulfate

o

l

-

-

0

1

3

1

Uronic Acid

Glucuronic acid

Glucuronic acid lduronic acid

Glucuronic acid Glucuronic acid (Galactose)

Glucuronic acid Glucuronic acid (?)

Amino Sugar

Glucosamine Galactosamine Galactosamine

Galactosamine Galactosamine

Glucosamin巴

Glucosamine

Glucosamine

Compound

Hyaluronic acid

CSA CSA-B (β-heparin) CSA-C

Chondroitin

Keratosulfate Heparin

Heparin-mono sulfuric acid

polysaccharide is devoid of anticoagulant properties. Chondroitinsulfuric acid-A

differs from chondroitinsulfuric acid-C in that the sulfate group is placed at position 4 rather than in position 626，2り Therehas been so far no evidence of differences in biological activity between these two compounds， both of which are devoid of effects on the antithrombic system. It is of interest， however， that chondroitinsulfuric acid-C seems to occur in large amounts in the tissues of the elasmobranchs28). This is of interest in that no ca1cification occurs in these cartilages. The possible rela-tionship of the position of the sulfate group with respect to ca1cification as well as the evolution of this difference in linkage， is particularly intriguing.

Chondroitinsulfuric acid-B represents a compound which is chemically quite differ-ent from chondroitinsulfuric acids-A and -C in that it contains L-iduronic acid in contrast with D-glucuronic acid found in the other two chondroitin sulfates. Meyer and his col1eagues originally found evidence that there occurs in skin a chondroitin sulfate different from that of cartilage. However， it was first demonstrated by Masamune and his colleagues that this probably contained a different uronic acid and this substance was called dermoitin sulfuric acid in their original studies. Subs-equent studies by Meyer and his colleagues and Dr. Cifonelli and myself， in our laboratory， showed that this compound contains L-iduronic acid29，30).

The next figure CFigure 5) shows the structure of L-iduronic acid compared with that of D-glucuronic acid. It will be noted that the two substances are similar except for the steriochemistry of carbon 5. L-iduronic acid is an epimer of D-glu-curonic acid. L-idose was not known to be present in natural material， but the derivative， L-idotol， was first found in mountain ash berry by de Bertrand31l， in

H

H O

COOH

H O

OH H OH H .. A comparison of the structure of L-iduronic and D-glucuronic acids.

L -IDURONIC ACID D-GLUCURONIC ACID

Fig. 5.

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1905. The uronic acid present in chondroitinsulfuric acid-B is， in bct， L-iduronic acid， and has now been established by a number of different techniques. Identifica-tion has been based on paper chromatography in various solvents and the conver-sion of the isolated L-iduronic acid to L-idonic acid， L-idotol， and a mixture of L-idose and L-idosan. These are summarized in the next figure (Figure 6).

yH20H

H-C-OH

HO-C-H

H-?一OH

HO-C-H

COOH

ー一一+

。同加 ?同，OH

H-C-OH H-C-OH

f一一一一C-H HO-C-H NABH. o H-C-OH 一一ームう H-C-OH

HH-H HO十 H

」一一一c、 CHρH

V L-IDITOL rL

M川

円V

Tl

pu AH

1」附

門

川

加

H加

H小川

xly--

一-o

fclCIG-FYAU--c

u

¥

一一--

4

H

O

H

O

L

E

M

H

U

H

えて¥

DJVE¥

《し‘、、A

R

C

M川

∞

1』

L-IDURONIC ACID

lMOHHCl NAsH. H+

。

τ山い」明

H¥?グO

H-C-OH

HO-?-H

H-C-OH

HO-C-H

CHpH

L-IDOSE

Fig. 6. Summary of reactions used to characterize uronic acid of β-heparin.

It is of interest that this substance was probably isolated by Marbet and Win-tersteinll2l from a commercial preparation of heparin and because of its anticoagulant properties was thought at that time to be an isomer of heparin rather than an isomer of chondroitin. Material supplied to us by Dr. Winterstein was used as a starting material for our structural studies.

Marbet and Winterstein pointed out that this substance has properties somewhat similar to heparin， yet di妊erentin that it is strongly antithrombic but much less anticoagulant than is heparin. More recently， Dr. Burton J. Grossman and myself have studied this reaction in much greater detail 33l • It was found出atthe anti-thrombic activity of s-heparin， or chondroitinsulfuric acid， varies with the concen-tration of thrombin. At low thrombin concentrations it is a more active antithrombic substance than is heparin， but at high thrombic concentrations it is inactive. Like heparin~. it requires for activity the thrombin cofactor， or the natural plasma anti-thrombin for its activity. In the absence of plasma it is completely inactive. The possible role that this substance may play physiological1y remains to be determined. On whole blood it has an anticoagulant activity of only 5 per cent that of heparin.

15

Despite the relatively low antithrombic activity the presence of large amounts in tissues might indicate that this substance plays a significant role in the normal homeostatic mechanisms which control the deposition of fibrin at a local area.

This substance has been found in other sites and was identified by Smith and Gallop34l in gastric mucosa and has been found in rabbit skin by Schiller ct al. 3日.

Dorfman and Lorincz36) pointed out that it is excreted in large amounts in the urine

of patients with the Hurler syndrome. This has been confirmed37l.・ Therole that it might play in the Hurler syndrome will be discussed in a later lecture.

Returning to Table L the polysaccharide， chondroitin， is of some interest. This was isolated by Meyer and coworkers38

) from cornea and was thought to be a desul-fated form of chondroitinsulfuric acid. It was suggested that this material might represent a metabolic precursor of chondroitinsulfuric acid. The recent findings of the uptake of radioactive sulfate as a result of the action of an enzyme present in cartilage has resulted in further suggestion that chondroition may， indeed， be a precursor39). However， the enzymes under study have not resulted net synthesis of polysaccharide.

Unlike other mucopolysaccharides， keratosulfate lacks the uronic acid group of polysaccharide. Instead a galactose group is substituted. Keratosulfate exists in the nuc1eus pulposus in high concentration. This is of interest in vi巴wof the embryonic derivation of this structure from the notochord. Recently， Shetlar and Masters40

)

and Kuhn and Leppelmann41l have shown that with increasing age there is an in-

crease in the ratio of glucosamine to galactosamine in cartilage. This finding has now found explanation in the work of Meyer ct al.42) who found that rib cartilage in the human adult contains a large proportion of keratosulfate in contrast to its absence in similar cartilage from newborns. Even larger amounts of keratosulfate were obtained from patients with the Marfan syndrome， although it is dubious whether this represented a significant difference.

Heparin monosulfuric acid is a compound which has only recently been studied in any detail. This substance was probably first isolated by Jorpes and Garde1l43l

in 1948 as a by-product from a commercial preparation of heparin. It was found to have many of the properties of heparin inc1uding a positive rotation， but in c

16

probably 1→6 glucuronido hexosamine. This substance has very little， if any，

antithrombic or anticoagulant properties. The origin of this substance and its meta-bolic importance has not yet been c1arified but its properties suggest that it may be related to heparin. It is possible that this may represent a metabolic by-product in the manufacture of heparin or an intermediate in the formation of that substarice. Perhaps the best known mucopolysaccharide is heparin， because of its great importance both medically and biologically. Athough this substance has been studied intensively from a biological point of view its chemistry is yet ill understood. It is known to contain more than 2 sulfate groups per disaccharide unit and to have a positive rotation. The exact structure of heparin is not yet c1ear. Unlike the other acid mucopolysac-charides it contains an N-sulfate group and no N-acetyl. In addition to the N-sulfate，

one、andone一halfsulfate groups are present per disaccharide rep巴atingunit.

Heparin is present throughout the organism in mast cells. Although it has been shown to have striking anticoagulant properties， its physiological role in the coagulation

mechanism requires further elucidation.

That the chondroitinsulfuric acid of cartilage is linked to protein was suggested as early as 1889 when Mδrner first obtained a substance which he called chondromucoid. Subsequent investigators indicated that chondroitinsulfate was linked to protein. The possibility that this represents a true compound was brought to the fore by the work of Shatton and Schubert山 whoisolated by low temperature extraction of cartilage a

material which they called “mucoproteinぺ Thismaterial has now been studied in great detail， primarily by Mathews40). He found that in cartilage there appears to be a protein complex which has a minimum molecular weight of approximately 4.0 X 106

with a length of approximately 3700 A. When the protein is destroyed， sodium chon-droitinsulfate is obtained with a molecular weight of approximately 50，000. This suggests that the protein molecule has chains of polysaccharide bound to it so that each linear sodium chondroitinsulfuric acid molecule is bound along the protein chain. It was suggested that approximately 62 units of chondroitin sulfate is present in each protein complex molecule. There was evidence that the protein complex associates to give molecules of mu

• ;冨 • • • • .図.;;Aimtyr-、...:1:. ... ;.晶.

'戸:?Ff・-ょ•• • ~.' . ..

•

• 直.

・・' ・-• •

• •

Fig. 7. A portion of the protein mucopolysaccharide complex. The center core represents the protein.

17

Thus. at high ionic concentration the neutralization of negative charges results in a decrease in mutual repulsion and an increased coi1ing of the polysaccharide chain. As the ionic concentration is decreased greater repulsion occurs and the chains are extend巴d.This results in a great increase in their viscosity. The exact role that this plays at specific localization of ions is as yet undetermined. However. it would seem reasonable 出atat certain areas. particularly. such as the condensation of polysaccharides at basement membranes. the presence of the highly charged long chain partic1es results in selective effects On the transport of ions.

1 have reviewed in this lecture certain aspects of the chemical composition of connective tissue and have attempted to consider this from the point of view of the functional capacity of these tissues. The specificity of certain components. their diversity. and yet their similarity. makes an intriguing chapter in the biology of tissues. The fact that all metabolizing cells of the higher organism are imbedded in a matrix containing these materials undoubtedly plays an important regulatory func' tion in the metabolism of tissues. It may be that by influencing the composition. both qualitative and quantitative. of connective tissue. certain agents such as nutri. tional factors and hormones are able to control the metabolism of tissues. Furthennore. the changes that occur in these tissues with progressive age undoubtedly play a role in the ageing process. 1n subsequent lectures we shall examine in more detai! the metabolism and biosynthesis of these substances and finally consider these from the point of view of the diseases of the connective tissue.

18

REFERENCES

1. Gross， J.， Sokal， Z.， and Rougvie， M.， ]. Histoche例. & Cytoche仇， 4， 227 (1956).

2. Maximow，A.， Mikr. Anat. For8ch.， 17， 629 (1925).

3. Kling，D.H.， Levine，M.G:， and Weiss，S.， Proc.50c.Exρtl. Biol. Med.， 89， 261 (1955).

4. Grossfeld， H.， Meyer， K.， and Godman， G.， Proc. 50c. E.ゆtl.Biol. Med. ， 88， 31 (1955).

5. Castor， C. W.， Proc. 50c. Exμ1. Biol. Med.， .94， 51 (1957).

6. Asboe-Hansen， G.， International Review of Cytology， 3， 399 (1954).

7. Ri1ey， James F.， in The Mast Cells， Wi11iams and Wilkins Co.， Baltimore， 1959. 8. Geiger， R.， Unpublished results.

9. Gustavson， K. H.， in The Chemistry and Reactivity of Collagen， Academic Press，

p.39， 1956. 10. Rich， A. and Crick， F. H. C.， in Recent Advances in Gelatin and Glue Research，

Pergamon Press， 1958.

11. Nageotte， J.， Comtt. rend. 80C. biol.， 96， 464 (1927). 12. Orekhovich， V. N.， Tustanovskii， A. A.， and Plotnikova， N. E.， Do!?lady Akad. Nauk

U55R， 60， 837 (1948). 13. Orekhovich， V. N.， Ustanovskii， A. A.， Orekhovich， K. D.， and Plotnikova， N. E.，

Biokhimiya， 13， 55 (1948).

14. Orekhovich， V. N. and Shpikiter， V. 0.， BiokM仰 iya，20， 438 (1955).

15. Gross， J.， Sokal， Z.， and Rougvie， M.， ]. Hi8tochem. & Cytochem.， 4， 227 (1956).

16. Gross， J.， ].Exρtl. Med.， 107， 247 (1958). 17. Gross， J. and Kirk， D.， ]. Biol. Chem.， 233， 335 (1958).

18. Gross， J.， ]. Exρtl. Med.， 108， 215 (1958). 19. Doty， P. and Nishihara， T.， in Recent Advances in Gelatin and Glue Research，

Pergamon Press， p.92 (1958).

20. Gardell， S.， Acta Chem. 5cand.， 9， 1035 (1955). 21. Dempsey，E.'再T.and Lansing， A. I.， International Revieωof Cytology， 3， 437 (1954).

22. Orekhovich， V. N. and Shpikiter， V. 0.， Biokhimiya， 20， 438 (1955). 23. Orr， S. F. D.， Harris， J. C.， and Synven， B.，肋ture，169， 544 (1952).

24. Banga， I.， Acta PhY8iol. Acad. 5ci.， Hung.， 3， 317 (1952). 25. Davidson， E. A. and Meyer， K.， ]. Am. Che刑. 50c.， 76， 5686 (1954).

26. Mathews， M. B.， Nature， 181， 421 (1958). 27. Hoffman， P.， Linker， A.， and Meyer， K.， Biochim. Bioρhy8. Acta， 30， 184 (1958)

28. Nakanishi， K.， Takahashi， N.， and Egami， F.， Bull. Chem. Soc.， ]aρan， 29， 434

(1956) . 29. Cifonelli， J. A.， Ludowieg， J.， and Dorfman， A.， ]. Biol. Chem.， 233， 541 (1958). 30. Hoffman， P.， Linker， A.， and Meyer， K.， Arch. Biochem. BioβhY8.， 69， 435 (1957).

31. de Bertrand， G.， Bull. 50c. ChI1n.， 33， 166 (1905).

32. Marbet， R. and Winterstein， A.， Heル.Chim. Acta， 34， 2311 (1951).

33. Grossman， B. J. and Dorfman， A.， Pediatric8， 20， 506 (1957). 34. Smith

19

43. Jorpes， J. E. and Gardell， S.， ]. Biol. Che制.， 176， 267 (1948). 44. Linker， A.， Hoffman， P.， Sampson， P.， and Meyer， K.， Biochi叩 . Bioρhys. Acta 189，

493 (1958). 45. Brown， D. H.， Proc. Natl. Acad. Sci.， 43， 783 (1957)

46. Schiller， S.， Biochem. Bioρhys. Acta， 32， 315 (1959). 47. Cifonelli， J. A. and Dorfman， A.， Unpublished results.

48. Shatton， J. and Schubert， M.， ]. Biol. Che制.， 211， 565 (1954).

49. Mathews， M. B. and Lozaityte，1.， Arch. Bioche叩• Bioρhys.， 74， 158・(1958).

50. Muir，H.， Biochem. ].， 69， 195 (1958).

20

2. Biosynthesis of Connective Tissue Components

Little is known regarding the mechanism of biosynthesis of connective tissue protein. Considerable effort has been focussed on the finding that proline is a more effi.cient precursor of the hydroxyproline of collagen than is free hydroxyproline 1，へA similar state of affairs exists for the relationship of lysine and hydroxylysine3，ベMore definitive knowledge regarding the mechanism of synthesis of these proteins awaits elucidation of the mechanisms of protein synthesis in general.

Before considering our knowledge regarding the metabolism of polysaccharides in connective tissue， we wi11 consider first in some detail the present state of knowledge regarding the biosynthesis of these substances. The pathway of biosynthesis of acid mucopolysaccharides is becoming rapidly c1arified. The metabolic study of connective tissue poses certain technical problems since， although connective tissues are present throughout the body， nevertheless their distribution is of such a nature that it is diffi.cu1t to obtain isolated connective tissues appropriate to biochemical study.

Because of this difficu1ty， about ten years ago we undertook to elucidate the mechanism of biosynthesis of acid mucopolysaccharides by employing a strain of Group A streptococcus. This is of convenience in that this organism produces large amounts of hyaluronic acid in its capsule and a considerable amount is excreted into the medium. Utilizing a standardized strain of this organism， it was possible to evolve conditions of study in which the origin of the fourteen unique carbon atoms could be determined. By utilizing specifically lab巴ledprecursors it was possible to establish that the glucuronic acid and the hexosamine moieties of hyaluronic acid are derived from glucose without scission of the carbon chain. These conc1usions were based on experiments utilizing both C -1 Iabeled and C -6 Iabeled glucose日.

Furthermore， it was possible to show that acetate could be directly utilized for the acetyl group of the N-acetylhexosamine. When more immediate precursors were studied it could be demonstrated that glucosamine did serve as a precursor， but n巴itherN-acetylglucosamine nor glucuronic acid were precursors for the formation of hyaluronic acid. Failure of glucuronic acid to serve as a precursor subsequently was cIarified by the discovery of the pathway of formation of glucuronides through the uridine nuc1eotides.

Ha

21

formation of phosphorylated glucose derivatives. It is established that fructos巴ー6-phosphate may be converted to glucosamine-6-phosphate by the transfer of the amide group of glutamine. It is not clear that this represents the sole pathway of glu-cosamine synthesis. The further pathway to the formatlon of N-acetylglucosamine-l-phosphate， which in turn reacts with uridine triphosphate to form uridine diphospho-N-acetylglucosamine， has been demonstrated. It has also been suggested by preli-minary experiments that the latter compound may be the precursor of the uridine diphosphomuramic acid which probably plays a role in bacterial cell wall synthesis山 .

GLUCOSE

↓仰GALACTOSE

1m

FRUCTOSE-6-P←ーGLUCOSE-6-Pー.，.GLLCO$E-¥平 GALACTOSE-1-P

↓…M ↓ヒミミJ↓山P

GLUCOSAM¥NE -6 -P UDPG一一主主ー令 UDPGι

1 ACETYL CoA ↓D附

N-ACETYLGLUCOSAM¥NE -6-P UDPGA

↓ j N-ACETYLGLUCOSAM¥NE -トP UDPGιA

↓仰L町

一

V

A

N--i山VU

G

M

p

p

n

u

n

U

H

U

H

U

Fig. 1. Pathway for the biosynthesis of uridine diphospho-N-acetylglucosamine and uridine diphosphoglucuronic acid.

Glucose-l-phosphate may traverse another pathway to form uridine diphospho-glucuronic acid， a compound which appears to be a key intermediate for many purposes. Oxidation of the 6 th carbon atom by widely distributed enzyme results in uridine diphosphoglucuronic acid. The latter compound may be converted to uridine diphosphogalacturonic acid. However， another pathway exists for the con-version of uridine diphosphoglucose to uridine diphosphogalactose. This pathway affords a mechanism for the interconversion of glucose and galactose in the body.

More recent studies have indicated that uridine diphospho-N-acetylglucosamine may be converted to the galactosamine derivatives13l

•

The two intermediates which are most1y likely candidates for the synthesis of an acid mucopolysaccharide may thus be formed by a series of known enzymatic reactions. The strong possibility that uridine diphospho-N-acetylglucosamine and uridine diphosphoglucuronic acid might be involved in the synthesis of hyaluronic acid led us to determine whether these intermediates do， in fact， occur in streptococci which synthesize hyaluronic acid. It was readily shown that both of these important nucleotides could be isolated from such microorganismsω.

More recent1y， we have been interested in the direct enzymatic synthesis of hyaluronic acid. In a series of studies out with Dr.恥1arkovitzand Dr. CifonelIi， we have obtained a cell-free preparation which synthesizes hyaluronic acid. A study

22

was facilitated great1y by the utilization of the technique of tritiation which had been described by Wilzbach1日 Thismade the treatment with gaseous tritium uridine diphosphoglucur・onicacid which had been isolated from another strain of streptococcus and obtain a labeled nucleotide. Our first experiments were carried out utilizing such labeled uridine diphosphoglucuronic acid combined with uridine diphospho-N-acetyl唱

glucosamine and N-acetylglucosamine士phosphate. When these compounds were mixed with an enzyme derived by a strain of streptococcus that had been used in our earlier studies， it was possible to isolate hyaluronic acid which showed radio-activity. Necessary for this reaction was the presence of magnesium. Such an ex司

periment is illustrated in Table 1. As will be noted the 0 time control showed little significant radioactivity while after incubation for 2 hours a significant amount of radioactivity was incorporated into the polysaccharide. On centrifugation of the enzyme， the activity could not be sedimented at 10， 000 x g but was sedimented at 100，000xg.

Table 1 Incorporation of H3 into HA from UDPGA.H3 by Sonicate of Group A Streptococcus

Time cpm per mg HA

O

2 hours

82

2550

AIl tubes contained 0.75 ml. of 15 min. sonicate of Group A streptococcus， μM of UDPGA-H"， UDPAG，

AG-1-P， ATP and 10μM of MgCl，・

UDPG A -H" == 580， 000 cpm/μM.

When several preparations labeled from UDP-glucuronic acid-H3 were repurified by adsorption on Dowex 1-Cl-(gX) and elution with 2 N NaCl， no significant change in radioactivity was evident. Analyses of one of the preparations showed a hexos-amine-uronic acid圃Nmolar ratio of 1. 00: O. 99: O. 94.

Further proof that the radioactive precursors were incorporated into hyaluronate was obtained by recovery of the glucosamine and of glucose derived from the glu-curonate portion of the molecule. As will be evident from subsequent experiments，

incorporation of tritium could be obtained from UDP-N-acetylglucosamine-H3 as well as from N-acetylglucosamine-1-P-H3.

The glucuronate moiety， after reduction to glucose， and the glucosamine moiety were isolated from hyaluronate labeled from UDP-glucuronic acid-H3， UDP-N-acetylglucosamine-H3， and N-acetylglucosamine-1-P-H3. The data shown in Table 2 indicate that the use of UDP-glucuronate-H3 as a precursor results in labeling ex-clusively in the glucuronic acid moiety. In contrast， the use of UDP-N-acetylglu-cosamine-H3 or N-acetylglucosamine士P-H3 as a precursor results in labeling in the glucosamine moiety with minimal labeling in the glucuronic acid moiety. The unex同

pected high specific activities of the glucose and glucosamine are unexplained. This may have resulted from variability of the counter since the hyaluronic acid was not counted at the same time as the degradation products.

A third method of verifying the incorporation of isotop巳 intohyaluronate depended upon enzymatic degradation. Hyaluronate preparations， labeled from GNAc-1-P-H3 were treated for an extended period of time with C. pC1.jトingen8hyaluronidase and the resultant unsaturated disaccharide 3-0-(s-D-Ll， 4， 5-g1ucoseenpyranosyluronic

Table 2. Isolation and Specific Radioactivity of Components of Differentially Labeled Hyaluronic Acid

Hyaluronic acid* Radioactivity

Tritiated precursor

UDP-GA

UDP-GNAc

GNAc-1-P

Radioactivity

cpm/ ，umole ***

460 740

2950 1550

1770 2660

Glucose料 Glucosamine

cpm/μmole cpm/μmole 。1510

82 1970

266 2650

* Hyaluronic acid was obtained in experiments with the 10，000 x g supernatant fraction.

紳 Derivedfrom the glucuronic acid of hyaluronic acid. *** Aμmole of hyaluronic acid is equated with a μmole of the disaccharide from

hyaluronic acid.

Table 3. Action of ClostridiaI Hyaluronidase on Hyalnronic Acid四H8

Tritiated precursor

UDP-GA UDP-GNAc GNAc-1-P

Total radio-Weight activity

mg. d.p.m.

20 72，000

10 130，000 10 922，000

Corrected Total radio-Corrected total Radio-total radio-activity of radio-activity activity activity H，0 of H，O in H，O

d.p.m. d.p.m. d.p.m. % 59，700 25，000 12，700 21.3 124，000 5，600 0 0 915，000 6，650 0 0

23

Each experimental tube contained the fol1owing per ml. of O.lM acetate:0.15M NaCl， pH 5.0; hyaluronate， 9.09 mg. ; clostridial hyaluronidas巴， 1.81 mg; penicillin， 2000 units. A control tube for each contained the above components without clos-tridial hyaluronidase. Incubation time was 19~告 hours at 370

• A 1.0 ml. sample from each was lyophylized and the water was assayed for radioactivity. The total radio-activity of water obtained by lyophylization from control tubes was subtracted from the total radioactivity of the hyaluronate and from the radioactivity of the water of the sample containing hyaluronidase. The percentage recovery of radioactivity in water was calculated from these corrected values.

acid)-2-deoxy-2-acetamido-D-glucose16) was chromatographed on paper with an acetic

acid-n-butanol-H20 (1:4:1) mixture. Elution of the disaccharide， as detected by

ultraviolet absorption. resu1ted in the recovery of 80 p巴rcent of the radioactivity of

the original hyaluronate. The ultraviolet spectrum at pH 2.0 was characteristic of

this unsaturated disaccharide.

Additional information was obtained from this type of experiment by determi-

nation of radioactivity in the H20. After enzymatic digestion， the H20 was recovered

by lyophilization at pH 5. 0 and the radioactivity determined as previously indicated.

The resuIts shown in Table 3 demonstrated that the H20 contains 21.3 per cent of

the radioactivity of hyaluronic acid-H3 derived from UDP-glucuronate-H3 (other com-

ponents are non-radioactive). Enzymatic hydrolysis of hyaluronic acid-H3 derived

from UDP-N-acetyIglucosamine-H3 and N-ac巴tylglucosamine-1-P-H3 released only small

amounts of radioactivity. Since the studies of Linker et al.16) indicate that bacterial

hyaluronidase produces unsaturation in the uronic acid portion of the disaccharide.

it was to be expected that hyaluronic acid synthesized from UDP-glucuronic acid-H3

24

would yield WOH when hydrolyzed by clostridial hyaluronidase. It is improbable that WOH would be rel巴asedby hydrolysis of hyaluronate labeled in the hexosamine portion of the molecule. The results of these experiments not only confirm the previous conclusions regarding the localization of tritium in various samples of hyaluronate， but lend support to the thesis of Linker ctαl. that unsaturation in the uronate moiety is produced by the action of bacterial hyaluronidases on hyaluronate. The relative ease of obtaining tritiated compounds15' combined with the use of en-zymes causing a similar unsaturation on other mucopolysaccharides17l indicate the possi-

bility of measuring polysaccharide biosynthesis by a very much simplified procedure. The data in Table 4 demonstrate an absolute requirement for UDP-N-acetylglu・

cosamine and MgCh and a relative requirement for N-acetylglucosamine-1-P and ATP. Since UDP-glucuronate-H3 was the source of radioactivity in these experiments，

the dependence on the presence of this compound could not be established. However，

Table 4. Requirements of Hyaluronic Acid-Synthesizing System

Tube No. UDP-GNAc GNAc-1-P 且在gCl， ATP Radioactivity of hyaluronic acid

μmole μmole μmole μmole cpm/mg.

1* 1.0 0.64 10 1.0 O

2 1.0 0.64 10 1.0 700 3 O 0.64 10 1.0 3 4 1.0 O 10 1.0 530 5 1.0 0.64 O 1.0 13

6 1.0 0.64 10 O 520

* Tube 1 was heated at the beginning of the experiment; all others were incubated at 370 for 130 minutes before heating. Cells were subjected to sonic disruption in 0.05 M PO.， pH 7.0， for 15 minutes

and centrifuged for 10 rninutes at 10，000 x g. All tub巴scontained 0.75 ml. of the supernatant enzyme in a total volume of 1.5 ml. including the following: P04， pH 7.0， 75μmoles; UDP-GA-H" (5.8x105 c.p.m.)，lμmole. An impure preparation of GNAc-l担Pwas used.

as illustrated in Fig. 2， wih N .acetylglucosamine-1司P-H3as a radioactive precursor，

there is a 6・foldincrease in radioactivity of hyaluronate with increasing concentrations of UDP-glucuronate. In other exp己rimentsthere was no incorporation of radio-activity frcim N-acetylglucosamine-1-P-H3 in the absence of added UDP-glucuronate. It is apparent that some extracts either contain or can form small amounts of the latter compound. Column chromatography of boiled extracts of this strain of strepto-coccus revealed relatively large quantities of UDP-glucuronate compared to UDP-N-acetylglucosamine14l. In view of the effect of pεnicillin on accumulation of uridine nucleotides 18ー加に an attempt was made to determine whether this antibiotic affects

hyaluronate synthesis. No effect was observed.

Fig. 3 demonstrates that Mg++， Mn+ヘor Co十十 activate the hyaluronate-synthesizing system to varying degrees. At lower concentrations than shown in Fig. 3， Mn++ is inactive although at 1μM it is more effective than Mg+ +. Addition of ethylenediaminetetraacetic acid in 3-fold mo1ar excess with respect to Mg+ + com司

p1etely abolishes hyaluronate synthesis. Dialysis of the enzyme atど resu1tedin marked 10ss of activity， which could be

prevented by the addition of O. 005 .M cysteine. Centrifugation for 1 hour at 105，400 x g

25

300

司工

否5 1.0 2.0

μMOLE UDPGA

Fig. 2. Effect of UDP-GA concentration on incorporation of GNAc-H3 from GNAc-1-P-H8 into hyaluronic acid (HA). The 10，000xg super-natant fraction was obtained as in Table 6 (time of sonic disruption， 30 minutes). All tubes contained 0.75ml. of the 10，000 x g supernatant fraction in a total volume of 1.5 ml. including the fol1owing in μmoles: PO.， pH 7.0， 75; cysteine， 3.75; MgCI2， 10; GNAc-1-P-H3， 0.097 (8.04 x 105 C. p. m.); UDP-GNAc， 1. 0; ATP， 1. O. Incubation time was 2 hours at 370

•

∞∞∞

円V

ハU

内

U

5

6

4

白

-U迂

O乏O広d

」4〉工由主

o M1cl2 ・MnCl2x CoS04

6000

10 20 30 40 50 60 下o 80 メIMOLEMETAI..++

Fig. 3. Effect of metal ion concentration on incorporation of GNAc-H3 from GNAc-1-P-H3 into hyaluronic acid. The 10，000xg supernatant fraction was obtained as in Fig. 2. Additions were the same as in Fig. 2， except 1μmole of UDP-GA was used and the metal ion con-centration was varied as indicated. Incubation time was 2 hours at 370

• The protein content of each tube was 2.9 mg.

results in the sedimentation of 85 per cent of the enzyme activity， indicating that

the major portion of the enzyme system is particulate_

Since absolute requirements for UDP-glucuronic acid and UDP-N-acetylglucosamine

were established， it was possible to test for the presence of enzymes involved in

the synthesis of these compounds.

Table 5 indicates that a combination of UDP-glucose and DPN replaces UDP-

glucuronate. The presence of UDP .glucose dehydrogenase is thus demonstrated.

This enzyme is not present in the washed particulate fraction obtained from the

10， 000 x g supernatant fraction. Previously this enzyme has been demonstrated in

26

Table 5. UDP-G lucose Dehydrogenase

UDP-GA UDP-G DPN Radioactivity of hyaluronic acid

μmole μmole μmole cpm/mg O O O 345 1.0 O O 5110 O 1.0 O 810 O 1.0 1.0 2970 O O 1.0 390

Cells were subjected to sonic disruption in 0.05 M PO.-O. 005 M cysteine-O. 025 M MgCl" pH 7.0， for 30 minutes and centrifuged for 10 minutes at 10，000 x g. All 、tubescontained 0.75 ml. of supernatant enzyme (4.35 mg. of protein) in a total volume of 1. 5 ml. including the following inμmol旬 P04，75; cysteine， 3.75; MgCl" 43.5; GNAc-l-P-H"， 0.097 (8.04x105 c.p.m.); UDP-GNAc， 1.0. Incubation time was 1 hour at 370

•

animals， plants， and bacteria21ー叩 Repeatedattempts to demonstrate UDP-glucose

pyrophosphorylase， utilizing glucose吐-P，UTP， and DPN to replace UDP-glucuronic

acid， were unsuccessfu1. Such resu1ts， together with the simu1taneous demonstration of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase

(below)， indicate the nonidentity of UDP-glucose and UDP-N-acetylglucosamine pyrophosphorylases in this organism.

Table 6 demonstrates that a combination of N-acetylglucosamine-1-P-H3 and UTP

replaces UDP-N-acetylglucosamine. Neither GTP nor CTP substitute for UTP. The

relatively high incorporation in this experiment without added UTP suggests that small amounts of UTP or UDP-N-acetylglucosamine were either present or could be

formed in this enzyme preparation.

Table 6. UDP-N-Acetylglucosamine Pyrophosphorylase

UTP CTP GTP Radioactivity of hyaluronic acid

μmole μmole μmole cpm/mg.

1.0 O O 6740 O 1.0 O 280 O O 1.0 300 O O O 350

Cells were subjected to sonic disruption in 0.05 M P04-O. 005 M cysteine， pH 7.0， for 60 minutes and centrifuged for 10 minutes at 10，000 x g. All tubes contained 0.75 ml. of the supernatant enzyme in a total volume of 1.5 ml. including the following inμmoles: P04， pH 7.0， 75; cysteine， 3.75; MgC12， 20; GNAc-l-P-H"， 0.097 (8.04 x 10' c. p. m.). GNAc十 P，0.64 (impure preparation); UDP-GA， 1. O. Incubation time was 2 hours at 370

•

Table 7 demonstrates that UDP and ATP substitute for UTP， in the absence of UDP-N-acetylglucosamine， indicating the pres巴nceof uridine nuc1eoside diphosphokトnase. This enzyme has previously been found in yeast24> and animal tissue24，25人These resu1ts suggest an explanation for the stimulation by A TP previously demon-

strated.

The results presented indicate that the supernatant liquid from a 10，000 x g sonic extract contains UDP-N-acetylglucosamine pyrophosphorylase. This enzyme has been

27

Uridine NucIeo田ideDiphosphokinase Table 7.

Radioactivity of hyaluronic acid

cpm/mg.

2140 4 2 8

450

ATP

μmole

O

O

O 1.0 1.0

UDP

μmole

O

O

1.0 O

1.0

UTP

μmole

1.0 O

O

O

O

Tube No.

ーム

ηLnJ44FU

The 10，000 x g supernatant fraction was obtained as in Table 5 (time of sonic disruption， 60 minutes). All tubes contained 0.75 ml. of the supernatant enzyme (2.20mg. of protein) in a total volume of 1.5ml. including the following inμmoles: PO.， pH 7.0， 75; cysteine， 3.75; MgCl" 43.5; GNAc-1-P-li'， 1.097 (8.04x1Q5 c.p.m.); UDP-GA， 1. O. Incubation tim巴 was1 hour at 370

•

demonstrated by Maley et al.26) and by Smith and Mills271 • The stimulation of

isotope incorporation by N-acetylglucosamine-l-P in the presence of UDP-N-acetylglu-

cosamine suggested the possibility of another role for the former. It should be noted

that N-acetylglucosamine was found to stimulate chitin synthesis in crude prepara-

tions28) and glucose was found to stimulate β-1， 3-g1ucan synthesis29).

i Radi…川hyaluronic acid

Exp.1 Exp.2

c.p.m./mg.

98 32

3

21

2060

2000

Table 8. Incorporation of GNAc-H8 from UDP-GNAc-H8 and GNAc-l-P-H3 into Hyaluronic Acid in Washed Particulate Prepar叫 ions

18

610

650

UTP

μmole

1.0

O

O

O

O

GNAc- GNAc-1-P-H3 1-P (8.0x10' c.p.m.)

μmole

1. 097

1. 097

1.097

O

O

μmole

O

O

O

1.0

O

UDP-GNAc-H3 (1.2 x 10' c. p. m.)

μmole

O

O

O

1.0

1.0

Tube UDP-No. GNAc

μmole

O

O

1.0

O

O

1

i

nノUHntυzA4Aphυ

The enzyme for Experiment 1 was obtained as follows: the 10，000 x g superna-tant fraction， obtained as in Table 6， was centrifuged for・ 1hourat 105，400xg (average)， the pellet taken up to the original volume in the same buffer solution in which the cells were disrupted， and recentrifuged for 1 hour at 105，400 x g. The pellet was washed a second time in this fashion and finally resuspended in the buffer so that 0.75ml. of the original 10，000 x g supernatant fraction was equivalent to 0.2 ml. of the washed pellet. The enzym巴 forExperiment 2 was that of Ex-periment 1 frozen， thawed， and washed two additional times. All tubes contain巴d0.2 ml. of the washed particulate enzyme in a final volume of 1.5 ml. including the following in μmoles; PO.， pH 7.0， 75; cysteine， 3.75; MgCl" 43.5; UDP-GA， 1. O. Incubation time was 1 hour at 370

•

In order to prepare an enzyme system free from UDP-N-acetylglucosamine

pyrophosphorylase， the active sediment obtained by high speed centrifugation was

washed repeatedly. The results of experiments performed with preparations obtained

in this manner are summarized in Table 8. The low incorporation from N-acetyl-

glucosamine士P-H3 in the presence of either UTP or UDP-N-acetylglucosamine

suggests that the enzyme responsible for formation of the latter has been largely， but not completely， removed. No stimulation by N-acetylglucosamine-l・P of incor-

28

poration of N-acetylglucosamine-Hs from UDP-N-acetylglucosamine-Hs was observed when this more purified enzyme was used. While it is possible that sufficient N-acetylglucosamine士P is produced by the remaining UDP-N-acetylglucosamine pyro-phosphorylase， or some other mechanism， the results of this experiment do not lend support to the hypothesis that N-acetylglucosamine-1-P plays some. role in hyaluronic acid synthesis other than as a precursor of UDP-N-acetylglucosamine. When the effect of UTP and pyrophosphate were studied with the particulate enzyme washed by centrifugation CFig. 4) it became apparent that both compounds inhibited the synthesis of hyaluronate despite the fact that UDP-N-acetylglucosamine pyrophos-phorylase was washed out of the preparation. In another experiment inhibition by UC>P was also demonstrated， but UMP had no effect.

280

Q ---5込 JU

i 160

室120

出

~ 80

40

• UTP o P-P

0.2 0.4 0.6 0.8 1.0 μM<コLE

Fig. 4. Inhibition of th巴 washedparticulate hyaluronic acid-synthesizing system by UTP and pyrophosphate (P-P). The enzyme was prepared as in Table 8， Ex-periment 1. All tubes contained 0.5ml. of the particu-late enzyme in a直nalvolume of 1.5 ml. including the following in μmoles: PO.， 75; cysteine， 2.5; MgCI" 50; UDP-GNAc-flS， 1.0 (1.2x105 c.p.m.); UDP-GA， 1.0. Incubation time was 2 hours at 370

•

Table 9 i1lustrates the results of experiments which demonstrate the net synthesis of hyaluronic acid by both unwashed and washed particulate preparations. Table 9，

Experiment 2， indicates that there was at least a lO-fold increase in nondialyzable uronic acid and hexosamine during the incubation. It is concluded that 90 per cent of the hyaluronate isolated from this preparation was synthesized 仇 vitro. Analyses of the purified hyaluronate of Table 9， Experiment 2， showed a hexosamine-uronic acid molar ratio of 1. 00 : 1. 10. From 50μmoles of UDP-glucuronate a theoretical recovery of 18.9 mg. of hyaluronate is to be expected. It can be ca1culated from the data in Table 9， Experiment 2， that a total net increase in nondialyzable uronic acid occurred equivalent to 7. 5 mg. of hyaluronate. Thus， 40 per cent of the available glucuronate became nondialyzable. Preliminary viscosity measurements by Dr. Martin B. Mathews， of this laboratory， indicate the molecular weight to be in the

range of 10， 000 to 50， 000.

29

Table 9. Net Synthesis of Hyaluronic Acid

Experiment Time Hyaluronic acid by

Acid albumin Uronic Acid Hexosamine

hr. mg. ロ19. 立19.

1 O 0.11 0.22 0.20

3. 5 0.80 0.67 0.53

ム 0.69 0.45 0.33

2 O 0.026 0.021

3.5 0.327 0.275

ム 0.301 0.254

Experiment 1: The particulate enzyme preparation was obtained as in Table 8. Experimen t 1. except tha t high speed cen trifuga tion was carried ou t a t 80.700 x g (average) and the high speed pel1et was not washed. Both tubes contained 0.5 ml. of the resuspended pel1et in a色nalvolume of 1.5 ml. including the fol1owing in μmoles: PO.. pH 7.0. 75; cysteine. 2.5; MgCl，. 62; UDP-GA. 2.0; UDP-GNAc. 2.0; GNAc-1-P. 2.0; ATP. 1.0. After the indicated incubation time at 370 both tubes were immersed in a boiling water bath for 5 minutes. cooled. brought to a volume of 4 ml.. centrifuged at 2000 r. p. m. for 20 minutes. the supernatant decanted. and the pel1et resuspended in 2 ml. of H，O and centrifuged as before. The supernatant and washings were combined and dialyzed against running tap H，O and then against distilled H，O. Analyses were mad巴 on the dialyzed preparations without further puri丑cation.

Experiment 2: The washed particulate enzyme preparation was obtained in Table 8. Experiment 1. One tube contained 12.5 ml. of the enzyme preparation in a final volume of 37.5 ml. including the following in μmoles: PO.. pH7. O. 1875; cysteine. 94; MgCl，. 1094; UDP-GA. 50; UDP-GNAc. 50; GNAc-1-P.20; ATP. 25. After the indicated incubation times at 37". 1. 5 ml. samples were removed and treated as in Experiment 1. Analyses were made on the dialyzed preparations without furth巴rpuri丑cationand are recorded in this table. The remainder of the incubated sample was boiled for 10 minutes and the hyaluronic acid isolated as described with the following exceptions: a) No carrier hyalUronic acid was added. b) After dialysis the material was concentrated to a volume of 5 ml. c) After the 宜nalpurification step indicated. the hyaluronic acid was passed over a 50 mg. Norit A-50 per cent stearic acid column. then over a Dowex 50-H十 column.and宣nal1ydialyzed.

Attempts to demonstrate a primer effect of either hyaluronate or testicular hyal-

uronidase treated material have been uniformly negative. Even in particulate prep-

arations that have been washed four times and on which analyses indicate O. 15μg. of nondialyzable uronic acid， no effect could be demonstrated of added hyaluronate

on incorporation of N-acetylglucosamine-H3 from UDP-N-acetylglucosamine-H3 into

hyaluronate.

DISCUSSION

Studies of disaccharide synthesis indicate that the glycosyl group is transferred

to acceptors which in al1 except one case Csucrose formation)3()) are phosphorylated

sugars. The known examples of disaccharide formation may be indicated as fol1ows:

UDP-glucose十fructose<:! sucrose + UDP

UDP-glucose + fructose-6-P <=主 sucrose-P+ UDP

UDP-glucose + glucose-6-P <=士 trehalose-6-P + UDP

UDP-galactose十glucose-1-P<=主 lactose-1-P + UDP

30

No mechanism has yet been suggested for the interaction of two uridine nuc1eo-tides to produce a disaccharide. If one assumes a mechanism for the initiation of polysaccharide synthesis simi1ar to that for disaccharide formation. it might be expected that some chain initiator is involved. As already indicated. studies with the crude enzyme system demonstrated a stimulation of hyaluronic acid synthesis by N-acetylglucosamine-l-P in the presence of UDP-N-acetylglucosamine. This led to the speculation that the former might be a chain initiator according to the following scheme:

UDP-GA十GNAc-l-P<=士 GA-GNAc-1-'P + UDP UDP-GNAc+GA-GNAc-I-P 己 GNAc-GA-GNAc-l-P+UDP UDP-GA+GNAc-GA-GNAc-I-P <=士 GA-GNAc-GA-GNAc-l-P+ UDP

This scheme envisages a unique disaccharide which is N-acetylhyalobiuronic acid phosphate to which are added alternate glucuronyl and glucosaminyl residues. Such a mechanism is attractive but the data in Table 8 indicate that stimulation of hyal・

uronic acid synthesis by N-acetylglucosamine士Pis lost on purification of the enzyme. A similar mechanism may involve primer (small molecular weight residues of hyal-uronic acid). We have thus far been unable to demonstrate any primer require-ment in this system although trace amounts of uronic acid containing material (as demonstrated by the carbazole method) are pr巴sentin the washed partic1es. It is of interest to note that primer requirements have been demonstrated in the cases of chitin， cellulose， and glycogen synthesis. It is possible that in the case of hyaluronic acid synthesis small amounts of chain initiators are tightly bound to enzyme.

Irrespective of the nature of the chain initiator. it is necessary to face the problem of mechanism in the alt巴rnation of groups. Previous studies31，32) have suggested that certain group transfers involve optical inversion. Thus it is postulated that those instances in which optical configuration appears to be retained represent two transfer reactions. the first involving binding of the glycosyl group to the enzyme，

and the second representing transfer from the enzyme to an acceptor. In other reactions. such as the phosphorolysis of maltose studied by Fitting and Doudoroff3日，

a single inversion occurs. It is assumed under these conditions that transfer occurs between substrates with no intermediate binding of glycosyl groups to the apoenzyme. If these considerations apply to the biosynthesis of hyaluronic acid from uridine nuc1eotides. certain expectations may be entertained. Although there is no direct proof that the nuc1eotides are α-linked. this assumption is supported by both chemical and enzymatic evidence. Since the linkages in hyaluronic acid are s. it might be expected that one (or three) group transfers are involved. A possible mechanism could then be indicated as follows (Fig. 5).

A single enzyme with three active sites is proposed. (A similar mechanism might involve two enzymes with a similar spatial relationship on a partic1e. Such a supposition would probably require different sites for the uridine portions of the two nuc1eotides). It is postulated that each of the uridine n

Fig. 5. Representation of a possible mcchanism of hyaluronic acid synthesis. The dark shaded balls J'cprcscnt glucuronic acid. the lighter balls N-acetylglucosamine. while the large ball represents UDPー Theblack bead represents an oxygen atom. See text for further explanation.

31

charide synthesis viαuridine nucleotides. Structl1re A in Fig. 5 indicates the state of the enzyme at the initiation of chain formation. X denotes a chain initiator which contains an N-acetylglucosamine moiety. (A similar mechanism may be written with glucuronic acid initially linked to the enzyme). In strl1cture A. UDP-glucuronic acid is bound to the' enzyme at sites 1 and 3. The initial reaction involves a formation of出eGA.→J X linkage by a transfer reaction involving rllpture of the anomeric C -0 bond of glucurouic acid. On the basis of Koshland's postulation. the resultant GA. →S

X bond will be β. conforming to the known structure of hyaluronic acid.

Following the formation of structure B. UDP dissociates fromせlemolecule as indicated in structure C.

Structure C now contains the beginning of hyaluronic acid chain held to enzyme bYLa glucuronic acid residue on site 3 while sites 1 and 2 are available for the binding of a molecule of UDP-N-acetylglucosamine. The resultant compound (structure D) 出enreacts to add an acetylg1ucosamine residue to the non-reducing end of the chain. yielding the β1→4 linkage known to exist in hyaluronic acid (structure E). The hyaluronic acid chain remains bound to the enzyme through the acetylglucosamine residue.

Such a mechanism not only explains alternation of groups but makes such orderly arrangement mandatory. Of particular interest is that such a mechanism could be

32

extended to explain the formation of more complex chains which contain more than two different monosaccharide units. The formation of s linkages is also explained.

Certain aspects of this mechanism are subject to experimental verification. It might be predicted that the addition of UDP-N-acetylglucosamine-H3 or UDP-glucuronic acid-H3 to the enzyme in the presence of UDP will not result in labeling of inactive UDP since no binding of N-acetylglucosamine or glucuronic acid to the enzyme(s) through C-l occurs. However， that one of the nuc1eotides， or both， is bound to the enzyme(s) is suggested by the finding that the enzymeCs) is stabilized in the presen-ce of the nuc1eotides and Mg+ +.

An alternative mechanism for the formation of hyaluronic acid might be the interaction of the two uridine nuc1eotides to form a disaccharide. The disaccharide must be linked to a phosphate or nuc1eotide to furnish energy for glycoside bond formation. Such a mechanism might be indicated as follows:

UDP-GNAc+ UDP-GA ー~ GNAc-GA-UDP+ UDP GNAc-GA-UDP+GNAc-GA-UDP→GNAc-GA-GNAc-GA-UDP+ UDP

This would require the involvement of two enzymes， one for the formation of the disaccharide uridine nuc1eotide and a second for polymerization of disaccharide units to the polymer. Again， provision must be made for inversion of the C -0 bond in the formation of the polysaccharide.

No critical evidence is yet available to choose between these mechanisms. The pathway through a disaccharide is less general since it is not applicable to synthesis of more complex polymers. Preliminary studies have revealed no evidence of the existence of intermediates in the reaction mixtures.

Thus far it has not been possible to solubilize the enzyme. However， treatment of streptococci with an enzyme obtained from Dr. Richard Krause of the Rockefeller Institute， results in the production of bacterial protoplasts. On osmotic lysis it was found that the protoplast membranes contain activity for the synthesis of hyaluronic acid.

The pathway of the biosynthesis of the sulfated mucopolysaccharide is not as yet c1ear. Incorporation of radioactive sulfate to chondroitinsulfuric acid by an enzyme obtained from epiphyses of chick embryo has been demonstrated釦. The participation of active sulfate， PAPS， apparently is similar to that in other sulfation reactions which have been elucidated. In a similar manner， Suzuki35) has shown iucorporati

33

acid-B was isolated. On degradation it could be determined that the iduronic acid contained a much higher radioactivity in carbon atom 6 than in the other carbon atoms， indicating that the L-iduronic acid derives from glucose without scission of the carbon skeleton. The mechanism by which this occurs is not clear. It is possible the epimerization of the 5th carbon atom occurs at the level of the uridine diphospho-glucose or the uridine diphosphoglucuronic acid， or may occur merely by conversion of glucose to idose by some other mechanism. In any case it is apparent that this represents a new type of epimerization of glucose.

Another important question regarding the synthesis of acid mucopolysaccharides involves the relationship of synthesis of the polysaccharide and protein moiety. It has been previously established， as indicated in the first lecture， that chondroitirトsulfuric acid-A in cartilage exists as a complex linked to protein. The existence of such a complex poses the question as to whether the fibroblast， the cell responsible for the formation of chondroitinsulfuric acid， forms the entire complex and extrudes this into the medium or rather forms the polysaccharide and the protein separately which are bound in some way in the extracellular substance. In order to answer this question it was decided to attempt to study the rate of turnover of the polysaccharide and the protein moiety. These studies were performed by Dr. John Gross in con-junction with Dr. Martin B. Mathews and myself.

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Fig. 6. The ratio of speci世cactivity at time (t) to that at (to) plotted against time on a semi-log scale.

Such a study could be carried out by specifically labeling the chondroitinsulfuric acid with sulfate and the protein part with l-carboxy labeled lysine. When such experiments were carried out it was possible to demonstrate that the rate of turnover of polysaccharide and protein moieties is the same， thus indicating that the entire complex is formed by the fibroblast. These data are i1lustrated in Figure 6. This finding is of considerable theoretical interest from the point of view of the overall metabolism of connective tissue polysaccharides and their role in health and disease and wi11 be discussed in a subsequent lecture.

34

REFERENCES

1. Stetten， M. R.， J. Biol. Chem.， 181， 31 (1949). 2. Wolf， G. and Berger， C. R. A.， J. Biol. Chem.， 230， 231 (1958).

3. Piez， K. A. and Likens， R. C.， J. Biol. Chem.， 229， 101 (1957). 4. Sinex， F. M. and Van Slyke， D. D.， J. Biol. Chem.， 216， 245 (1955). 5. Dorfman， A.， Markovitz， A.， and Cifonelli， A.， Federation Proc.， 11， 1093 (1958).

6. Leloir， L. F.， Arch. Bioche叩.Bio戸hys.，33， 186 (1951).

7. Leloir， L. F. and Cabib， E.， J. A仰.Chem. 50c.， 75，5445 (1953).

8. Leloir， L. F. and Cardini， C. E.， f. Biol. Chem.， 214， 157 (1955).

9. Leloir， L. F. and Cardini， C. E.， J. A仰• Chem. 50c.， 79， 6340 (1957). 10. Dutton， G. J. and Storey，1. D. E.， Biochem. f.， 57， 275 (1954).

11. Cabib， E.， Leloir， L. F.， and Cardini， C. E.， J. Biol， Che叩.， 203， 1055 (1953).

12. Strominger，J.L.， Federation Proc.， 17， 318 (1958).

13. Glaser， L.. J. Biol. Che刑.， 234， 2801 (1959). 14. Cifonel1i， J. A. and Dorfman， A.， J. Biol. Che仰.， 228， 547 (1957).

15. Wilzbach， K E.， J. A叩• Chem. 50c.， 79， 1013 (1957). 16. Linker， A.， Meyer， K.， and Hoffman， P.， J. Biol. Che刑.， 219， 13 (1956). 一

17. Hoffman， P.， Linker， A. ， Sampson， P. ， Meyer， K. ， and Korn， E. D. ， Biochim. et Bioρhys. Acta， 25， 658 (1957).

18. Park， J. T.， J. Biol. ぐhem.，194， 877 (1952).

19. Park， J. T.， J. Biol. Chem.， 194， 885 (1952).

20. park， J. T.， f. Biol. Che叩.， 194， 897 (1952). 21. Strominger， J. L.， Maxwell， E. S.， Axelrod， J.， and Kalckar， H. M.， J. Biol. Chem.，

224， 79 (1957). 22. Strominger， J. L. and Mapson， L. W.， Biochem. f.， 66， 567 (1957). 23. Smith， E. E. B.， Mi11s， G. T.， Bernheimer， H. P.， and Austrian， R.， Bioche叩. et

Bioρhys. Acta， 28， 211 (1958).

24. Berg， P. and Joklik， W. K.， J. Biol. Chem.， 210， 657 (1954). 25. Krebs， H. A. and Hems， R.， Biochi1n. et Bioρhys. Acta， 12， 172 (1953).

26. Maley， F.， Maley， G. F.， and Lardy， H. A.， f. Am. Chem. 50c.， 78， 5303 (1956). 27. Smith， E. E. B. and Mills， G. T.， Biochi.例 . et Bioρhys. Acta， 13， 386 (1954).

28. Glaser， L. and Brown， D. H.， f. Biol. Che仰.， 228， 729 (1957).

29. Feingold， D. S.， Neufeld， E. F.， and Hassid， W. Z.， J. Biol. Chem.， 233， 783 (1958).

30. Cardini， C. E.， Leloir， L. F.， and Chiriboga， J.， f. Biol. Chem.， 214， 149 (1955). 31. Fitting， C. and Doudoroff，1¥ι， f. Biol. Chem.， 199， 153 (1952). 32. Koshland， D. E.， Jr.， Biol. R仰s.，28， 416 (1953).

33. Bucher， T.， Biochim. et Bioρhys. Acta， 1， 292 (1947). 34. Gross， J.1.， Mathews， M. B.， and Dorfman， A.， Federation Proc.， .18， 239 (1959).

35. Suzuki， S.， Federati・onProc.， 18， 1327 (1959).

36. Korn， E.， J. Bi

35

3. Metabolism of Mucopolysaccharides in Connective Tissues

In previous lectures we have considered the general problems. of connective tissue and the mechanisms of biosynthesis of mucopolysaccharides. However. an understanding of the physiological role of these substances requires a more complete understanding of the detail of the factors which control both the qualitative and quantitative changes in the metabolism of these substances. A wide range of studies has now appeared which have been concerned with those of radioactive sulfate to identify mucopolysaccharides in connective tissue and to determine their rate of metabolism. Radioactive sulfate has the great advantage of being relatively in-expensive. easy to handle. and it specifically labels ester sulfate groups. Since the acid mucopolysaccharides represent the largest group of ester sulfate-containing compounds in the body. this method has found very great usefulness. However. it is limited by the fact that it does not determine the rate of metabolism of hyaluronic acid and the carbon skeleton of other acid mucopolysaccharides. Earlier studies mentioned in lecture 2 furnished information regarding the precursors of the carbon skeleton of the acid mucopolysaccharides.

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This information provided a background for choosing appropriate precursors for study of rates of metabolism of mucopolysaccharides in mammalian tissues. Skin was selected since sufficient material could be obtained for isolating the individual mucopolysaccharides. Methods" were devised for the isolation of pure acid muco-polysaccharides. Ground defatted skin is extracted with 2% NaOH. After removal of NaOH. the extract is treated with trypsin followed by precipitation with trichloro-acetic acid. A crude polysaccharide mixture is precipitated from the supernatant with ethanol， and flnal purification is achieved by slab electrophoresis on celite. This procedure resu1ts in a sharp separation of the sulfated and nonsulfated polysac-charides. A typical pattern of separation is illustrated in Fig. 1. Table 1 shows the detailed analyses of the two fractions. The agreement with theory for each

36

fraction is excellent except that the uronic acid value is low for the sulfated fraction as determined by the carbazole method in contrast to a theoretical value obtained by the C02 method. This anomalous behavior has been shown to be due to the presence of L-iduronic acid in chondroitinsulfuric acid同B2，3lin contrast to D-glucuronic

acid in hyaluronic acid and chondroitinsulfuric acid-A. Chondroitinsulfuric acid-B，

unlike chondroitinsulfuric acids-A and -C， is completely resistant to testicular hyal-uronidase. The sulfated mucopolysaccharide of skin is partially hydrolyzed by testi-cular hyaluronidase， suggesting a mixture of chondroitinsulfuric acid-B， and chon-droitinsulfuric acid-A. In the metabolic studies to be reported this mixture was not resolved so that the data represent metabolic properties of the mixture. The hyaluronic

手cidfraction behaved in all respects like that obtained from other sources.

Table 1. Chemical Analyses of Mucopolysaccharide Fractions from Rabbit Skin

Figures represent per cent

Theoryh) RHA-1 % of Theory

Theoryil RCSA-1 % of Theory

Loss on dryinga)

O 8.5

O 11.3

Nb) Sc) Hexuronic H~~~}:~nic acidd) acide)

Hyaluronic Acid Fraction 3.49 O 48.4 48.4 3.18 くO.1 44.3 46.6 91.1 91.5 96.3

Chondroitinsulfuric Acid Fraction 2.78 6.37 38.6 38.6 2.75 6.03 35.7 14.3 98.9 94.7 92.5 37.0

Hexosaminef) N-acety1g)

44.7 10.72 39.0 10.00 87.2 93.2

35.6 8.55 32.1 7.92 90.2 92.7

a) Loss at 780 in uacuo over P，05 for 60 hrs. All other ana1ytica1 va1ues in this tab1e are corrected for this 10ss.

b) By micro-Kje1dahl. c) By turbidimetric ana1ysis following Carius combustion仙.

d) By manometric CO， method31'.

e) By co10rimetric carbazo1e reaction'2'. f) By a modification of the E1son-Morgan method33' following hydro1ysis in 4NHC1

for 14 hrs. at 1000•

g) By chromic acid oxidation8山 .

h) Theory for disaccharide repeating unit， C14H20011NNa. i) Theory for disaccharide repeating unit， C14H19014NSNa，

Normal rates of metabolism of acid mucopolysaccharides of skin were studied first4，引 Reportsfrom the laboratories of Bostromト 9) and Dziewiatkowski10) have shown that S35 administered as inorganic sulfate is incorporated into sulfated acid

mucopolysaccharides. Such studies， however， do not permit any conclusion regard-ing the rate of metabolism of the carbon skeleton and may measure only the rate of a sulfate exchange reaction. For these reasons experiments were carred out with C14-containing precursors. In later investigations radiosulfate was also used simultaneously. Experiments were performed on both rabbits and rats. Acetate-l-C14 injected into rabbits was found to be a precursor of mucopolysaccharides. It was immediately apparent that hyahironic acid is metabolized at a more rapid rate than chondroitinsulfuric acid. Degradation of the skin polysaccharides shows that when acetate-l・C14is administered， the polysaccharides are labeled solely in the acetyl group. Since it is possible that incorporation of radioactivity results from an exchange reaction of the acetyl group and therefore is not necessarily a measure of mucopolysaccharide synthesis， glucose-U-C14 and S3504一orcarboxyl-labeled acetate

37

and S35U4--were used simultaneously in later experiments. The results of a typical experiment utilizing glucose-U司C14and S35U4--are shown

in Fig. 2. Each of eight rabbits CSwift strain) received 49μc of glucose-U-Ct4 and 1. 2 mc. of Na2S35u4. une pair of rabbits was sacrificed 16 hours after the last in-jection and the remaining six rabbits were killed in pairs， 4， 8， and 16 days after administration of isotopic precursors. This experiment was complicated by the pro-bability that the 16・hourinterval before sacrifice of the first group .of animals was not sufficient to obtain maximal labeling. This period had been found adequate in previous experiments with acetate. The difference may by due to the slower absorp-tion of glucose. The activity at the time of maximal labeling， therefore， was ob-tained from the intersection of the uptake and decay curves. Despite departure from linearity， the data depicted in Fig 2 demonstrate that C14 is lost from hyaluronic acid more rapidly than from chondroitinsulfuric acid. The half-life times as calculated from the line of best fit， are 3.7 days for hyaluronic acid and 7.7 days for chon-droitinsulfuric acid.

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Fig. 2. A plot of the C14 of the hyaluronic acid (HA) and chondroitinsulfuric acid (CSA) of the skin of rabbits (corrected for body weight) 吋ecetdwith C14-g1u・cose and Na，S'50.・Zeroday on the abscissa indicates the time of the last injection. The ordinate represents the log of the radioactivity in counts per minute (CPM) corrected to "in丑nitethickness". Maximal radioactivity was obtained by extrapolation， as described in the text.

Fig. 3. A semilogarithmic plot of the C14 of glucosamine compared with that of the hyaluronic acid (HA) isolated from the skin of rabbits iniected with C14-gtucose and Na，S"O.・ Thedata are plotted by a line of best fit calculated by the method of least squares from the values obtained at 3， 7， and 15 days. The values for maximal labeling were obtained by extending the line to the ordinate. The C14 was measured as BaC03， corrected to“in世nitethickness".

The two mucopolysaccharide fractions were hydrolyzed， the hexosamines were isolated from both the hyaluronic acid and chondroitinsulfuric acid， and the sulfate from the chondroitinsulfuric acid. Fig. 3 illustrates that the radioactivity of the glu-cosamine parallels that of the hyaluronic acid andthat the C14 concentration of the glucosamine is similar to that of th巴 entiremolecule. The findings are in keeping with the concept that both the glucosamine and glucuronic acid portions of the molecule derive from glucose. More direct evidence for this has recently been furnished by the use of glucos-6-C14 11l.

38

Fig. 4 i1lustrates simi1ar results for the chondroitinsulfuric acid fraction except that the rate of turnover is also indicat巴dby the sulfate group. The slope of the radioactivity V8. time for the whole polysaccharide， the galactosamine， and the

sulfate are all reasonably equal. The half-life time of chondroitinsulfuric acid cal-

culated from the curves was found to be 7.7 and 10.0 days， respectively， on the basis of the rates of disappearance of C14 and S35.

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Fig. 4. A comparison of the decay curves for the C14 of galactosamine and chon. droitin.sulfuric acid (CSA) and for the S.5 of the chondroitinsulfuric acid isola. ted from the skin of rabbits injected with C14・glucoseand Na，S.50，・ Thedata are plotted by a 1in巴 ofbest丑tcalculated by the method of least squares from the values obtained at 3， 7， and 15 days. The valu巴sfor maximal labeling were obtained by extrapolating to zero time. The C14 and S85 were measured as BaCO. and BaSO.， respectively， corrected to“in宜主litethickness".

Fig. 5. A semilogarithmic plot of the C14 of the hyaluronic acid (HA) and chon-droitin'sulfuric acid (CSA) and of the S.5 of the chondroitinsulfuric acid isolated from the skin of rats injected with C14.carboxY1.1abeled acetate and Na，S.50，・Each point represents the value obtained from a pool of twelve rat skins. The data are plotted by the method of least squares. The C14 and the S.5 were measured as BaCO， and BaSO.， respectively， corrected to “infinite thickness".

That the turnover of the entire polysaccharide was measured found confirma-tion in subsequent experiments employing acetate-1-C14 and Na2S"O.・ Eachrat was

injected with 57μc. of acetate-1-C14 and 15μc. of Na2S"O. as an isotonic mixture. The animals were sacrificed in groups of twelve 1， 3， 5， 9， and 17 days after an injection of isotopes. A portion of the results of this experiment is illustrated in Fig. 5. The data indicate， once again， the marked difference in rates of disap-

pearance of C14 from hyaluronic acid and chondroitinsulfuric acid. The similarity of the slopes for S35 and Cl. in chondroitinsulfuric acid confirms that these measure-

ments truly refiect the rate of metabolism of the entire polysaccharide molecule and validate experiments of Bostriim and Gardell剖.

Repeated experiments have now b巴enperformed estimating the rates of meta-

bolism of both hyaluronic acid and chondroitinsulfuric acid. Some of these are

summarized in Table 2. The difference between the two polysaccharides is consis-tent1y observed in both rats and rabbits whether acetate or glucose is used as pre-

cursor. It should again be emphasized that the chondroitinsulfuric acid fraction represents a mixture of two different polysaccharides.

39

Table 2. Compari目。nof Half-Life Times of Skin Mucopolysaccharides and Some of Their Component自制

Hyaluronic acid (HA) Chondroitinsulfuric ac函 (CSA)Species C14-HA N-Ac巴削 ，C:~:c_o; I CH-CSA N-Acetyl 9:~:t;>- S" samlne sa立llne

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The development of methods and the determination of parameters of normal metabolism have permitted an investigation of the effects of hormones on the meta-bolism of acid mucopolysaccharides in skin. The tedious nature of the methods has limited the number of investigations that so far could be undertaken. We are able at present to report on the effects of insulin1Z，l3>， hydrocortisonel4l and certain pre-liminary observations concerning the effects of growth hormone.

The possibility that insulin is involved in metabolism of acid mucopolysnccharides was suggested by the finding that both the glucosamine and uronic acid portions of the molecule derive from glucose. Since insulin is involved in the utilzation of glucose and since the early steps of mucopolysaccharide biosynthesis and other pathways for the utilization of glucose are probably identical， it seemed reasonable to postulate a role for insulin in the biosynthesis of these compounds.

Diabetes was induced in rats by a single subcutaneous injection of 150 mg. of al-loxan monohydra:te per kilo of body weight. Animals that did not lose weight were eliminated. Three weeks after the administration of the alloxan， blood glucose determined on rats selected at random ranged from 410 to 592 mg. per 100 ml. In treated animals. insulin was injected subcutaneously in daily doses of 20 or 40 iinits per kilo of body weight. This dose was varied because continued administration of the higher dose resu1ted in evidence of hypoglycemia in some animals. During insulin treatment. glucose levels ranged between 25 and 45 mg. per 100 ml. of blood.

Sixty rats were divided into 3 experimental groups of equal size. une group of animals was used 3 weeks after the administration of alloxan. A second group served as untreated controls. while a third group of normal animals was maintained on half the average daily food intake for 3 weeks prior to. and during. the experi-ment. The weight loss of the latter group was similar to that of the diabetic animals. Each of the 60 rats was injected once subcutaneously with 80μc. of acetate-1・C14and 2.7μc. of NazS35u.. as an isotonic mixture. Ten rats in each group were sacrificed 1 and 5 days after the injection.

Fig. 6 represents the isotope concentration of the mucopolysaccharides isolated from the skin of these groups of animals. The diabetic animals show a striking decrease in the uptake of CJ. by the hyaluronic acid and of C14

40

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Fig. 6. A comparison of the C" concentration of hyaluronic acid and of the C" and S35 concentration of chondroitinsulfuric acid among the 3 experimental groups following the administration of acetate-l-C" and Na2S"O.・Theleft司 andright-hand bars of each pair repre-sent the values 1 and 5 davs， respectively， after the injection of isotopes. The value of the chondroitinsulfuric acid-C" at 5 days is omitted in the case of the diabetic rats. See text for explanation.

since the radioactivity of this sample was too low for accurate counting. While half司

life times ca1culated from two points are not accurate， the values serve to indicate gross changes in turnover. The apparent half帽lifetimes of 2. 6 days for hyaluronic acid and 11.0 and 10.8 days for chondroitinsulfuric acid Cbased on the C14 and S35，

respectively) found in the skin of normal animals， agree with those obtained previously from more detailed decay curves4，15l. In the diabetic animal， however， the turnover is considerably slower as evidenced by an apparent half司lifeof 4. 5 days for hyaluro-nic acid and 20.9 days for chondroitinsulfuric acid Cbased on S35).

The result of this experiment indicated a decreased capacity to metabolize acid mucopolysaccharides in diabetic animals. A fall in concentration of these substances might therefore be anticipated. Since methods for isolating the mucopolysaccharides are not quantitative， attempts were made to estimate possible changes in mucopoly-saccharide concentrations by utilizing the method of isotope dilution. The results，

indicated in Table 3， demonstrate a marked decrease in hyaluronic acid concentra司

tion and a less striking decrease in chondroitinsulfuric acid concentration. These determinations of pool size permitted the ca1culation of the turnover rates presented in Table 3. The difference between diabetic and normal animals is evident. Re-cently we have been able to show by a direct analytical technique that the quantity

Table 3. Cornparison of Pool Size and Turnover Rate in Norrnal and Diabetic Rats

Pool size") Turnover rat巴blMucopolysaccharide Subject (mll'/100 gm. ) (mg"/100 gm/day)

HA Normal 215 58

HA Diabetic 88 14

CSA Normal 187 12

CSA Diabetic 145 5

a) Pool size as determined by isotobe dilution method and expressed as mil1igrams per 100 gm. of acetone-defatted skin.

、 Poolsize b) Turnover rate=一一一一一一一一

ノ t1/2 X 1. 44

41

of acid mucopolysaccharides in the skin of alloxan diabetic rats is diminished. The sizes of the hyaluronic acid and chondroitinsulfuric acid pools in the skin

of the fasted animals were not measured， so that turnover rates comparable to those calculated for the normal and diabetic rats could not be obtained.

As already indicated， the use of acetate-1-04 effects specific labeling of the N-acetyl component of the mucopolysaccharide molecules. Since acetate utilization is decreased in the diabetic animal16，ベ itmay be argued that the observed resu1ts mi-rror alterations in acetate metabolism. Although the similarity of the 04 and S35 data would appear to validate this objection， another experiment was undertaken utilizing glucose-U-04 as well as Na2p3504・ Inthe same experiment the effect of insulin on both normal and diabetic rats was studied. Four experimental groups we-re used. At appropriate times， as designated below， each animal received a single subcutaneous injection of an isotonic mixture containing 6. 7μc. of glucose-U-C14 and 13.3μc. of Na2S350.・ Twogroups were made diabetic as described above. Three weeks later 50% of the diabetic animals were injected with the radioactive mixture. The remaining animals were treated daily with 20 or 40 units of insulin per kilo of body weight for 1 week before the administration of the radioactive ma-terial and daily thereafter until sacrifice. The two other groups of animals consisted of nondiabetic rats. One group served as a normal control while the other was inje-cted daily with 20 or 40 units of insulin p巴rkilo of body weight， before and after receiving the isotopes.

Eight to ten rats in each group were sacrificed at intervals of 1， 5， and 17 days after the administration of radioactive material， and the hyaluronic acid and chond-roitinsulfuric acid fractions were isolated from the resp巴ctivepools of skin.

CI4

1000

80C

三ι

0600

400

200

HA CSA

cl4

口NORMALllllI NORMAL-INS.Rx ・DIABETIC図 DIABETIC-INS.Rx

Fig. 7. A compari邑onof the CH concentration of hyaluronic acid and of chondroitinsulfuric acid of the various groups 1 (Ieft bar) and 5 (right bar) days after the administration of glucose-U-C14 and Na2S350.・

A comparison of the left bars of the diabetic and normal groups (Fig. 7) indi-cates a marked decrease in incorporation of isotope in both the hyaluronic acid and chondroitinsulfuric acid. This finding is entirely in accord with the results obtained when acetate-1-Cl.l was employed as a precursor. Calculation of half-life times for hyaluronic acid again showed some prolongation in diabetic animals (5.0 days compared with 3.8 days for normal) although these differences were not as striking as those observed for chondroitinsulfuric acid or those obtained in the previous experiment.

42

2 535 I!li NORMAL-IN5.Rx ・DlABETIC

園 DIABETIC-INS.Rx

Fig. 8. A comparison of the S35-concentration of chondroitin-sulfuric acid of the various groups following the administra-tion of glucose-U-C14 and Na，S3504. The bars in each group. reading fron left to right. represent values 1. 5. and 17 days after the injection of the isotopes.

The sulfate data CFig. 8) demonstrate. as in the previous experiment. a marked inhibition of isotope uptake in diabetic animals， a1though the differences in decay Chalf-life times) are not as evident.

The administration of insulin to diabetic animals restores the defect of uptake toward normal as illustrated by the data for C14 in hyaluronic acid CFig. 7) and for both C14 and S35 in chondroitinsulfuric acid CFigs. 7 and 8). The half-life times were actually shorter than normal in this group CHA-04>， 1. 9 days; CSA-Cl4>， 3.9 days; and CSA-S35， 6.3 days). This is not surprising since these animals were hy-poglycemic. The effect of insulin in normal rats was somewhat variable and not as striking as in the diabetic animals， probably because the diabetic animals are more

sensitive to insulin. In the present study an attempt was made to relate the pancreas to the synthesis

of acid mucopolysaccharides. The data show that in diabetic rats the uptake of C14 by the hyaluronic acid and of C14 and S35 by the chondroitinsulfuric acid is dimini-shed， the sizes of the mucopolysaccharide pools in the skin are decreased， and the hyaluronic acid and chondroitinsulfuric acid turnover rates are slower. In the insulin-treated diabetic animal， the metabolism of the skin mucopolysaccharides assumes a more normal character.

The radioactivity at zero time of the hyaluronic acid and chondroitinsulfuric acid fractions isolated from the skin of the diabetic animals was approximately one-third that of the same fractions isolated from the skin of the normal animals when either acetate-l-C14 or glucose心-04 and Na2S3504 were administered. The finding that the uptake of the isotopes was reduced to the same extent with each precursor used con-firms and supports the conclusion that the various components of the respective acid mucopolysaccharides turn over at the same rate. Furthermore， the observations argue against the view that an increased initial dilution due to larger than normal body pools of acetate and glucose is responsible for the resu1ts in the diabetic animal. It is unlikely that the ac巴tate，glucose， and sulfate pools were increased to exactly the same extent. The resu1ts of this investigation indicate that the synthesis of the connective tissue mucopolysaccharides is inhibited in the insulin-deficient animal.

43

More recently we have found that the method of determining the rate of turn-over has certain difficulties implicit in it. These include the laborious nature of the experiments plus the difficulties of accurate measurement of the pool size. For this reason we have instituted a new group of studies which depend on a more quantita-tive determination of the amount of acid mucopolysaccharides in connective tissue. The development of a new method for the determination of acid mucopolysaccharides is based on the differential solubility of complexes formed with cetyl pyridinium chlor-ide and is based on the original abservations of Scott山 .

Utilizing this method it is possilbe to separate quantitatively on a microscale the mucopolysaccharides of skin. When this was applied to rat skin a surprisingly large amount of .heparin was found. The data are illustrated in Table IV. Most noteworthy is the marked decrease in the concentration with increasing age.

The effects of adrenocortical hormones on connective tissues are so widely re-cognized that it seems unnecessary to review in detail th巴 extensiveliterature. The specific mechanism， however， by whichthese hormones affect connective tissues are not clear. Layton19

' found that sulfate fixation in the skin of intact rats is inhibited by cortisone administration. Autoradiographs show a considerably smaller S35 content in the skin of cortisone-treated rats than in skin of normal rats引.

Table 4. Effect of Age on Concentration of Mucopolysaccharides in Rat Skin

Age (days)

21* 23* 44* 57* 74* 217* 406 473 561 893 951

Hyaluronic Acid Chondroitinsulfuric Acid

μgm. uronic acid/gm. dry rat skin 979 385 894 406 1232 337 439 218 540 197 578 273 320 140 450 200 480 257 380 135 538 256

Heparin

421 562 388 100 92 111 70 110 68 35 100

* These data were obtained from pools of 8 to 10 rat skins for each age group. In the older age groups， the resu1ts r巴presentvalues from individual animals.

The results of Layton 19' and of Bostrδm and Ode blad 9九 whichare based on the use of S35， imply that the formation of sulfated mucopolysaccharides is inhibited in the skin of cortisone-treated animals. Cortisone has also been reported to inhibit the incorporation of sulfate and thereby increase the relative concentration of nonsulfated mucopolysaccharides in syphilomas of rabbits20

'. This conclusion is based solely on the weights of the mucopolysaccharides isolated by a technique which yields question-

able recoveries.

Bostrom and Odeblad9' demonstrated an inhibition of S35 uptake by the chon司

droitinsulfuric acid isolated from cartilage slices after incubation with cortisone. The in vitro response of cartilage slices to cortison巴 andcortisone ac巴tatewas confirmed by Clark and UmbreitzυThese investigators found that the addition of hydrocorti-sone or its acetate ester to the in Vit1'O system enhanc巴dthe uptake of S35 by car-tilage and concluded that thein vitro response of cartilage to adrenal steroids

44

appeared to be unrelated to their physiological action. The report that cortisone acetate has no effect while free cortisone increases the 'in vitro uptake of S35 by

granulation tissue of guinea pigS22) corroborates such a view.

Because of these uncertainties regarding the action of adrenal cortical hormones on connective tissue polysaccharides， experiments were conducted to investigate further

this question. The effect of cortisone acetate on the uptake of isotopes following ad-ministration of acetate-1・C14 and S3504 -- was studied. Four days of treatment with

5 mg. of cortisone acetate daily produced a moderate decrease in isotope uptake. These resu1ts suggested a more detailed experiment on the effect of duration of treat-

ment with adrenocortical hormones.

、Sev巴ntyrats were injected subcutaneously with 62. 5μc. of acetate-1・C14 and 30

μc. of Na2S3504 as an isotonic mixture. One group of 10 rats was sacrificed 24 hours

later. The radioactivity of the hyaluronic acid and chondroitinsulfuric acid isolated

from the pool of 10 rat skins represents the uptake of untreated animals at zero time. At this time daily subcutaneous injections of 5 mg. of crystalline hydrocortisone ace-

tate (suspended in saline-Tween 80) per animal were started. Three groups of 10

rats received the adrenal steroid and the remaining 3 groups of 10 rats serv巴d as controls. One group each of treated and untreated animals were sacrificed 3， 5， and 9 days， respectively， after administration of the radioactive mixture and the hyaluro-

nic acid and chondroitinsulfuric acid were isolated from each group comprising a pool

of 10 rat skins.

2，3

S35

肘γOROCORT1S0NE

些迦& d4

8

5

0

内

〆

』

内

ζ

〉ト一〉一↑

U4U一L一U凶止的

hL000」

HYORCニORT!50NE

仏、

Jザ

ρ

9

8

7

S

:

2

I

l

l

-

寸

-U〉ピ〉一トudu-LG凶仏的

LOUOJ

2

4 DAYS

'"'12 2'るう"巧

Fig. 9. Fig. 10.

Fig. 9. A semilogarithmic plot of the C14 of the hyaluronic acid isolated from the skin of normal rats (0) and of rats treated with hydrocortisone (x). The decay curve of the hyaluronic acid from the normal animals was obtained by the method of least squares. The curve of the hyaluronic acid from the ani-mals treated with hydrocortisone acetate was drawn through the experimentally determined points. Injections of hydrocortisone acetate were started at zero time as described in the text.

Fig. 10. A semi10garithmic plot of the C14 and 835 of the chondroitinsulfuric acid isolated from the skin of normal rats (0) and of rats treated with hydrocorti-sone acetate (x)， The decay curves for chodroitinsulfuric acid from the normal rats were obtained by the method of least squares. The curv巴sof th巴

chondroitinsulfuric acid from the animals treated with hydrocortisone acetate were drawn through the experimentally determined points. Injections of hy-drocortisone acetate w巴restarted at zero time as described in the text.

4 DAYS

CHONDROITIN SULFURIC ACID

1.50 1.5

1Iρ

45

The apparent half-life times in the skin of the normal rat， as ca1culated from the lines of best fit， are 3.1 days for the hyaluronic acid (Fig. 9) and 7.2 and 5.3 days for the chondroitinsulfuric acid on the basis of the C14 and S35 decay rates， res-pectively (Fig. 10).

When treatment with hydrocortisone acetate is initiated at the time of maximal labeling， it is possible to demonstrate a gradual decrease in the rate of turnover of the mucopolysaccharides in the skin (Figs. 9， 10). The inhibitory eff，ect of the steroid is manifest after 4 days of treatment and becomes more pronounced by the end of the experimental period.

The results of this study confirm and extend earlier findings9，19) regarding an inhibition by cortisone of S35 uptake in the chondroitinsulfuric acid of skin. Cortisone and hydrocortisone appear to depress not only the incorporation of sulfate in chond司

roitinsulfuric acid， but the metabolism of mucopolysaccharides in skin. The striking effects of growth hormone on cartilage have suggested that the

hormone has a specific effect on prolifetation of this tissue and may have an effect on the biosynthetic processes concerned with maintaining its matrix. A few scattered reports have attempted to correlate the effect of growth hormone on cartilage with the metabolism of chondroitinsulfuric acid. These studies depend upon the incorpora-tion of radiosulfate into cartilage and are based on the findines of Bostrom6) and Dziewiatlωwski23) that S35， administered as Na2S35u4， appears in the cartilage primarily as chondroitinsulfuric acid in 24 hours.

Ellis et al. 24) demonstrated a decreased uptake of S35 by costal cartilage of hypo-physectomized immature rats and a restoration toward normal fol1owing administration of growth hormone. Similar changes were described by Denko and Bergensta125九who reported that tibial caps and xyphoid cartilage appeared to be less sensitive to hy-pophysectomy and growth hormone than costal cartilage. An effect of growth hormone on the uptake of S35 by cartilage of normal rats could not be demonstrated. However， Murphy et al. 26)， in accord with previous observations made by Dziewiatkowski271 found that in normal rats the uptake of S35 by the proximal end of the tibia decreased with increasing age and body weight.

The studies of Murphy et al.2引 alsoshow a rapid decline following hypophysec-tomy in the uptake of S35 by the proximal tibial epiphyses of rats. A minim

46

acetate-1-C14 003μc.) and Na2S250. 00μc.) as an isotonic mixture. The animals

were sacrificed in groups of six 1. 3， 5， and 9 days after the injection. A group of

unoperated Iittermates was similarly tr巴ated. Growth hormone， 100 p，g. per rat， was

injected daily for 8 days in hypophysectomized animals， starting 3 weeks after hypo-

physectomy. On the 8th day of growth hormone treatment， the mixture of labeled

acetate and suIfate was administered and the animals were sacrified 1， 3， 5， and 9

days later. Daily injections of growth hormone were continued until the animals were kiIled.

c'4 o-NORMAL ・-HYPOX Cl4 o-~心RMAL

O-HYPOX 20 x-G.H

~ 1.6 u E 的 111

8 -'

1.2

I.OL O 2 4

DAYS Fig. 11.

8

Fig. 12

Fig. 11. A semilogarithmic plot of the CH of the hyaluronic acid isolated from the skin of normaI rats， hypophysectomiz巴d rats， and hypophysectomized rats treated with growth hormone. The curves were obtained by the method of Ieast squares.

Fig. 12. A semiIogarithmic plot of the C14 of the chondroiti'nsuIfuric acid fraction isolated from the skin of normal rats， hypophysectomized rats， and hypophy田

sectomized rats treated with growth hormone. The curves were obtained by the method of least squares

S35

3.5

トE〉 五3

定

量 3.1

出8

27

O 2 4 DAYS

o-NORMAL '-HYPOX X司 G.H.

8

Fig. 13. A semilogarithmic plot of the S35 of the chondroitinsulfuric acid fraction isolated from the skin of normaI rats， hypophysectomized rats， and hypophysectomized rats treated with growth hormone. The curves were obtained by the method of least squares.

The decay curves， based on disappearance of C14 and S35 from the mucopoly-

saccharides in the 3 experimental groups， are illustrated in Figs. 11. 12， and 13.

47

They demonstrate a marked decrease in disappearance of isotopes from both hyaluronic acid and chondroitinsulfuric acid following hypophysectomy. Only the turnover of the chondroitinsulfuric acid fraction appears to be restored to normal by growth hormone administration.

HA CSA

22

口NI.コRMAL・HYPOX~H-GH

DAYS AFTER HYPOPHYSECTOMY

Fig. 14. Uptake of C14 by hyaluronic acid and by chondroitinsulfuric acid in normal rats. hypophysectomized rats. hypophysectomized rats treated with growth hormone. Three groups of hypophysec' tomized animals are included -5. 15. and 22 days after hypophy' sectomy. respective]y. Growth hormone was administered daily beginning 15 days after hypophysectomy.

The uptake of 04 by bo出 hyaluronicacid and chondroitinsulfuric acid 24 hours after an injection of acetate-1・Ct4decreases with time after hypophysectomy (Fig. 14). when measured 5. 15. and 22 days postoperatively. Growth hormone (100 J-Lg. per rat per day)， administered from the 15th to the 22nd day has no effect on hyaluronic acic but restores the uptake of 04 by chondroitinsulfuric acid toward normal. These data in skin agree with the reported effect of growth hormone on S35 fixation by cartilage in the hypophysectomized rat. Contrary to the finding in cartilage26)， the uptake of C14 by the mucopolysaccharides continues to fall progressively during the posthypophysectomy period.

These results parallel those obtained from the decay curves. While hypophy-sectomy appears to decrease the turnover of both hyaluronic acid and chondroitinsulfuric acid， growth hormone restores only chondroitinsulfuric acid toward normal. That the effect of growth hormone is confined to the sulfated mucopolysaccharide may be significant in designating to this compound a sepcific function in cartilage and bone.

Final conclusions regarding the effects of hypophysectomy and growth hormone on turnover rates and net synthesis must await adequate measurement of pool sizes.

The physiological and pathological effects of hormones are undoubtedly the results

48

of infIuences on many metabolic processes. It would be simple if these effects could be explain巴dby a single fundamental reaction affecting different cell types and their functional expressions. The rapid progress in enzyme chemistry出athas helped to unravel the complex pathways of intermediary metabolism has created an expectation that similar reactions will completely elucidate hormonal effect. However， other mechanisms should not be overlooked. Regulation by endocrine agents is characteristic of multicellular organisms. 1n such organisms， the individual cells live in an envi-ronment which is governed by complex mechanisms. Is is not improbable that varia-tions in milieu profoundly affect the metabolism of al1 cells. Studies on the nature and the factors which control and regulate this milieu are still in their infancy. Even basic interactions between cell membrane and its environment are， as yet， poorly comprehended.

Recent studies have established that connective tissues are intricate systems containing many highly secific substances. The approach discussed today is limited in scope， since only gross changes in mucopolysaccharides were investigated. These investigations， however， demonstrated profound effects by the hormones on rates of metabolism. Other effects on mucopolysaccharides may be of equal or greater signi-ficance. Nothing is known of more subtle alterations on molecular size， molecular shape， or interaction with proteins.

Specific effects have been established nevertheless and open up new pathways of investigation. The mechanism which insulin influences mucopolysaccharide metabolism is not cl巴ar. The r巴sultsmay merely refl巴ctthe action of insulin on glucose utiliza-tion. Present evidence suggests出atformation of these heterologous polysaccharides proceeds by way of uridine nucleotides， although there is no conclusive evidence for this. Whatever the fundamental mechanism may bε， the defect in mucopolysaccharide metabolism is an expression of insu1in deficiency. The failure of wound healing and susceptibility to infection in diabetes mellitus may be a demonstration of the distorted mucopolysaccharide metabolism. Of particular interest for further investigation is出epossible role of this defect in the genesis of vascular degeneration.

There seems no question that cortisone and hydrocortisone infiuence a large

49

cells of the synovial membrane form hyaluronic acid. The effects presented in this

paper are limited to those of skin. It is possible that if connective tissues in other

areas had been studied， quite different results might have been obtained. In an early

experiment on the chick comb29ヘnoinhibition by cortisone of hexosamine accumula-

tion could be demonstrated， although testosterone caused a marked increase in hexo-

samine. In the same animals， testosterone appeared to have no effect on the hexo-

samine concentration of skin from other parts of the body.

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