THE RELATIONSHIP OF EPINEPHRINE AND GLUCAGON TO LIVER PHOSPHORYLASE

II. ENZYMATIC INACTIVATION OF LIVER PHOSPHORYLASE*

BY WALTER D. WOSILAIT AND EARL W. SUTHERLAND

(From the Department of Pharmacology, School of Medicine, Western Reserve University, Cleveland, Ohio)

(Received for publication, June 16, 1955)

The inactivation of liver phosphorylase in slices or extracts is catalyzed by an enzyme which was designated liver phosphorylase-inactivating en- zyme (1). The inactivated phosphorylase formed in slices could be reac- tivated following addition of epinephrine or glucagon. In order to study the inactivation of purified LP1 the inactivating enzyme was purified to a degree adequate for these studies. The soluble purified enzyme, on incu- bation with LP, caused a release of inorganic phosphate coincident with the loss of phosphorylase activity (2). The release was measured chem- ically in TCA supernatant solutions of enzymatically inactivated LP, and also by radioactivity when the substrate was radioactive phosphorylase (3). Ultracentrifugal analysis of LP and inactive LP showed that the two forms had the same sedimentation constant. The term inactive LP was used since activity was not restored when tested in the presence of 5-AMP, another example of differences existing between phosphorylase forms in liver and muscle. This paper deals with the purification, properties, and mode of action of the inactivating enzyme from liver.

Methods

Materic&-The liver phosphorylase used routinely as a substrate in these studies was the third alcohol fraction (4) which had been dialyzed overnight against water. These preparations were stable for months at -20” and withstood repeated freezing and thawing. For the large scale inactivations, dialyzed samples of LP of Step 9 (4) were used. In some cases these were precipitated with 2 volumes of cold neutral Versene-treated ammonium sulfate previously saturated at room temperature. The precipitate was dissolved in cold water, centrifuged for 40 minutes at 100,000 X g, repre-

* This research was supported in part by grants from the National Science Founda- tion (No. G-660) and Eli Lilly and Company.

1 The following abbreviations are used: LP, dog liver phosphorylase; IE, liver phosphorylase-inactivating enzyme; Tris, tris(hydroxymethyl)aminomethane; G-l-P, glucose-l-phosphate; 5-AMP, adenosine-5-phosphate; TCA, trichloroacetic acid.

469

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470 INACTIVATION OF LIVER PHOSPHORYLASE

cipitated as above, dissolved in a minimal amount of cold water, and di- alyzed. The specific activity of these samples varied between 20 and 31.

Glycogen, G-l-P, Tris, ammonium sulfate, and calcium phosphate gel were prepared as described in Paper I (4). The ammonium sulfate solu- tions used were saturated at room temperature and neutralized with con- centrated NH40H. Sodium sulfate was recrystallized from an aqueous solution saturated at 45” containing 0.6 gm. of disodium Versene per liter. After standing overnight at O”, the crystals were washed successively with ethanol, ethanol and ether (1: l), and ether. The dried crystals were heated for about 24 hours at 110”.

Caffeine and 5-AMP were purchased from the Nutritional Biochemicals Corporation. Phosvitin was purchased from the Worthington Biochemical Corporation. The samples of (Y- and fl-casein were generously supplied by Dr. T. I,. McMeekin of the Eastern Regional Research Laboratory.

Analytical Methods-The analytical procedures used were as previously described (4), except that a number of analytical procedures were adapted to 0.5 ml. systems and absorptions were measured in Pyrocells in a Beckman model DU spectrophotometer. Among the procedures adapted were phos- phorus determinations by the methods of Fiske and Subbarow (5) and Lowry and Lopez (6), pentose with the use of orcinol (7), carbohydrate with anthrone (8), glucose according to Nelson (9), and ashing of samples with perchloric acid as described by Bessey et al. (10).

Tissue Xamples-Samples for the enzyme survey were obtained from a young male dog which had been anesthetized with ether and bled by sever- ing the neck vessels. Samples of various tissues were placed in test-tubes, frozen immediately in a dry ice-alcohol bath, and then stored at -20” until ready for assay. In most cases the extracts for the survey were prepared by grinding samples of the tissues in a cold TenBroeck glass homogenizer with 10 to 20 volumes of cold water. The protein concentration of the various homogenates for the survey was determined by the method of Weichselbaum (11).

Standard Assay-The activity of IE was determined by measuring the amount of LP inactivated during a 10 minute incubat,ion with IE at 37”. The reactions were started by the addition of 0.2 ml. of IE (about 1 y of purified enzyme) to a mixture at room temperature containing 0.5 ml. of 0.1 M Tris (pH 7.2), 0.2 ml. of caffeine (1.0 mg. per ml.), approximately 0.75 unit of LP, and water to a final volume of 1.5 ml. After a 10 minute incu- bation at 37”, 0.5 ml. of a solution containing 5 per cent G-l-P, 1.5 per cent glycogen, and 0.3 M NaF (adjusted to pH 6.1 with HCl) was added and the residual LP was determined by measuring the rate of liberation of phos- phate from G-l-P. After an additional 10 minutes at 37”, a 0.5 ml. aliquot of the reaction mixture was transferred to cold TCA and the amount of

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W. D. WOSILAIT AND E. W. SUTHERLAND 471

phosphate present in an equivalent of 0.1 ml. of reaction mixture was determined. The per cent of LP inactivated was determined by the dif- ference from a corresponding control in which IE was omitted; values were used when the extent of inactivation was less than 65 per cent. The rate of inactivation with purified IE was almost linear to 50 per cent inactiva- hion; however, correction factors were used in the 50 to 65 per cent range in order to convert to linearity; for example, the factor at 55 per cent was 1.05, at 60 per cent 1.10, and at 65 per cent 1.15. 1 unit of inactivating enzyme was defined as that amount which catalyzed the inactivation of 1 unit of LP in the above assay. The specific activity was expressed as units per mg. of protein.

Results

Purijication Procedure

Steps 1 and g-The first two steps of purification were the same as those used in purifying LP (4). The animal was sacrificed, the liver was perfused with fluoride, and, after homogenization in a Waring blendor, a filtrate was obtained by use of a Hormann filter press. The specific activity of the homogenate varied between 1.1 and 1.6, while that of the filtrate was about 2. The inactivating enzyme was adsorbed on calcium phosphate gel as described and the supernatant solution was used as a source of phosphorylase. These and all subsequent steps were carried out at O-3” unless specified otherwise.

Step 3. Gel Washes and El&on-The gel was washed twice with 0.02 M Tris (pH 8.0) containing 10-3 M MgCL (1 liter per 300 t.o 400 gm. of liver), then washed three times in a similar fashion except that the buffer was at pH 9.0. The last wash was 0.5 the volume of the previous washes in order to collect the gel in one centrifuge bottle. Glass-distilled water was used routinely, and gel collections were made by 10 minute centrifugations at 500 X g.

Elution was carried out at 30” for 10 minutes, a volume of a solution of 0.05 M Tris (pH 10) and 0.3 M Na2S04 equal to one-half the weight of the tissue being employed. The eluted gel was collected by centrifugation at 7000 X g at room temperature for 10 minutes and discarded. The clear yellow eluate was chilled in an ice water bath and adjusted to pH 8.0, as measured with the glass electrode, with 1 N acetic acid. Adsorption of t.he enzyme on the gel, combined with extensive washing and subsequent elution, resulted consistently in a 40-fold or greater purification. While purification at this step was consistently good, the yields were variable.

Step 4. First Ammonium Sulfate Fractionation-Ammonium sulfate pre- cipitates were collected after waiting for 10 minutes by centrifugation

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472 INACTIVATION OF LIVER PHOSPHORYLASE

for 10 minutes at approximately 7000 X g. $ volume of saturated ammo- nium sulfate was added to the eluate. The enzyme was precipitated from the supernatant solution by adding 23 gm. of ammonium sulfate and 1 ml. of 1 N KOH per 100 ml. of eluate. The precipitate was dissolved in 0.1 M Tris (pH 10) by using 3.0 ml. per 100 gm. of tissue. This step re- sulted in almost 2-fold purification.

Step 5. Second Ammonium Sulfate Fractionation-A fraction was re- moved by the addition of 6/11 volume of saturated, Versene-treated am- monium sulfate. This step was somewhat variable and often an additional drop or 2 of ammonium sulfate were required to get the slight desired haze. The solution was centrifuged and the precipitate was discarded. The supernatant solution was made more alkaline by the addition of 0.5 N KOH (2 ml. per 17 ml. of solution) and was brought to 50 per cent saturation with saturated ammonium sulfate. The precipitate was col- lected and dissolved in 1 ml. of 0.2 M Tris (pH 9.0) for testing. The en- zyme was quite stable when lyophilized and lyophilization was carried out after the addition of 2 ml. of 0.2 M Tris (pH 9.0) per ml. of enzyme solution. Specific activities of this fraction ranged between 200 and 300 with about 10 per cent recovery of the total activity in the homogenate.

Step 6. Ethanol Fractionation of Lyophilisate-The powder was stable for weeks when stored in the cold under vacuum and portions could be purified further when needed. For example, 18 mg. of powder were dissolved in I.5 ml. of 0.05 M Tris (pH 8.0) and 1.5 ml. of 1 per cent NaCl. The enzyme was precipitated by adding 1.5 ml. of 80 per cent ethanol (-20”). The solution was allowed to stand for 7 minutes in an ice water bath and was centrifuged for 7 minutes at approximately 1000 X g at O-3”. The precipitate was dissolved in 1 ml. of 1 per cent NaCl. Such preparations were generally used, but often these samples were heated for 15 minutes at 50” before use. Fractionation with ethanol often resulted in a 2-fold purification with 50 to 70 per cent recovery of activity. These preparations were soluble and the activity remained in the supernatant solution when centrifuged for 40 minutes at about 75,000 X g.

Distribution of Enzymes-The results of a survey of various dog tissues are shown in Table I. The analyses were carried out under standard conditions with and without the presence of 0.1 M NaF during the incuba- tion of LP with the tissue sample. Inhibition by fluoride was taken to be indicative of the presence of an inactivating enzyme (or enzymes) similar to the one present in liver. Pancreatic extracts, for example, resulted in complete inactivation of LP even in the presence of fluoride, presumably due to the action of proteolytic enzymes. In blood an inactivating en- zyme was found to be present in the red blood cells while little or no in- activating enzyme was found in the plasma.

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W. D. WOSILAIT AND E. W. SUTHERLAND 473

Properties of IE

Stability of Enzyme-The condition found most suitable for storage of the purified enzyme was the lyophilized state and little activity was lost over a period of weeks at 0” in VOXUO. The enzyme, after fractionation with alcohol, usually stored well at 3” for a day or 2 in relatively high concentrations of NaCl and could be incubated at 50” for 15 minutes to inactivate contaminating enzymes, such as small amounts of pyrophos- phatase, with little or no loss of IE activity. The enzyme was not stable in a dilute state for extended periods and all activity determinations were

TABLE I Distribution of Inactivating Enzymes in Dog Tissues

Tissue I

Specific activity*

Liver . . . . . . . . . Kidney. . . . . . . . . Spleen . . . . Pituitary. Base of brain. Brain cortex. Lung. Skeletal muscle. . . Heart. Bladder. Intestine. Intestinal mucosa. Femoral artery..

.

.

.

.

1.2-1.6 1.2 0.5 0.4 0.1 0.1 1.0 0.3 0.3 0.8 0.4 0.6 0.4

* See the text for unit of measurement.

made just after dilution in 0.005 M Tris (pH 7.2). Enzymatic activity was lost rapidly below pH 5.5.

pH Optimum-The effect of pH on the enzymatic inactivation of LP was determined by substituting 0.01 M buffers for 0.1 M Tris (pH 7.2) in the standard assay. This lower buffer concentration was chosen so as to avoid pH changes in the final analysis of phosphorylase activity. The optimum for activity was near pH 8, as shown in Fig. 1; stability measure- ments accompanying the measurement of the pH optimum showed that this was not a result of an increased stability of the enzyme. Glycyl- glycine and Tris buffers served equally well, Verona1 and glycine were less satisfactory, while citrate and pyrophosphate were unsuitable since they inhibited 70 per cent or more at 1c2 and 1e3 M, respectively.

Efect of Substrate Concentration-Since the substrate molarity was very low (7 X 10v8 M), it was of interest to determine whether IE was saturated

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474 INACTIVATION OF LIVER PHOSPHORYLASE

at this low level. Standard assays were carried out, except for varying the initial LP concentrations and making appropriate dilutions of the reaction mixtures to carry out assays for the residual phosphorylase. It was found that IE was not saturated with substrate in the amounts used in the standard assay or by 10 times that amount of LP.

Effect of Various Compounds on Rate of IE Activity-The effect of various ions on IE activity was studied by separate additions to the standard assay. At the same time the effect on controls containing LP

PH FIG. 1. Effect of pH on IE activity. The standard assay was used with 0.01 M

buffer as indicated in place of 0.1 M Tris. @ = acetate, X = Tris, 0 = glycylgly- tine, A = imidaaole.

and no IE was studied. At final concentrations of 5 X 10Y4 M, Na+, K+,

and Ill@+ had no influence on the rate of the enzymatic reaction, while at final concentrations of 5 X lo+ M and lower, Hg++, Al+++, and Zn++ markedly decreased IE activity. A number of salts used in purification exerted inhibitory effects at concentrations of 0.013 M and greater, e.g. NaCl, Na2S04, and (NH&SOI. NaF was found to be an inhibitor of IE and 90 to 100 per cent inhibition was approached when the final concen- tration was 0.01 M. A number of other substances tested for their effects on the reaction included Versene, glutathione, and cysteine which ap- peared to have no significant effect at 10e3 M concentration.

The purified enzyme was inhibited by 5-AMP and inosine-5-phosphate, as noted previously when crude enzyme was studied (1). The activit,y of

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W. D. WOSILAIT AND E. W. SUTHERLAND 475

the purified enzyme on liver phosphorylase was stimulated by the addition of caffeine and theobromine, as reported previously (I), and also by menadione.

Products of Enzymatic Inactivation of LP

Formation of Inorganic Phosphate-With purified preparations of in- activating enzyme and liver phosphorylase, experiments were performed in order to determine whether some small group might be formed during the inactivation reaction. Since the substrate in these reactions was of high molecular weight, it was necessary to use relatively large amounts of the dialyzed enzyme (200 to 500 units of phosphorylase purified through Step 9 (4) or further). Phosphorylase was incubated at 25” for 30 to 60 minutes with and without the addition of IE protein usually in amounts varying between 0.5 and 2.5 per cent of the amount of LP protein. The pH of the reaction was determined by that of the two protein solutions used and was usually about 7.3. Following incubation, some inactivated samples were dialyzed versus small volumes of water and the dialysates were compared with those from controls. In most cases inactivated samples were treated with TCA or perchloric acid and, after centrifuga- tion, the supernatant solutions were compared with those from controls. A comparison of dialysates or TCA supernatant fluids of phosphorylase with and without enzymatic inactivation revealed no differences in amounts of pentose, ultraviolet-absorbing material, phenolic type compound, or peptide. Inorganic phosphate, however, was formed from LP during inactivation and, as reported earlier, the time-course of liberation of phosphate from LP was coincident with the loss of LP activity (2). Table II summarizes an experiment in which the amount of phosphate released paralleled the loss of enzymatic activity. It can be seen that negligible amounts of TCA-soluble phosphate are released from LP or IE alone and that release of phosphate from the two together ceases when inactivation ceases. The amount of phosphate liberated was essentially the same when measurements were made for stable phosphate or for phosphate as deter- mined by the method of Fiske and Subbarow or by the procedure of Lowry and Lopez. From ten experiments the amount of phosphate lib- erated was calculated to be 0.3 y of P per mg. of LP (on the basis that 30 units of LP equal 1 mg. of LP) or about 1 mole of phosphate per 100,000 gm. of enzyme. This appears to represent about 25 per cent of the total phosphate present in these preparations as determined by ashing (10). Whether this extra phosphate is related to LP or to a contaminant is not yet clear, but treatment with Norit or incubation with ribonuclease does not appreciably lower the phosphate content, nor is the extra phosphate released on prolonged incubation with IE as shown in Table II;

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476 INACTIVATION OF LIVER PHOSPHORYLASE

The formation of TCA-soluble phosphate during inactivation was also measured by the use of isotopes. Purified radioactive (P”“) phosphorylase was prepared for use as a substrate, as described in Paper III (12), by incubating liver slices with P32-orthophosphate and epinephrine. Table III summarizes an experiment in which chemical measurements of TCA- soluble phosphate were accompanied by measurements of isotopic phos- phate in both TCA supernatant fluids and precipitates. It can be seen that the radioactive phosphate (which had been incorporated into LP ill slices) remained with the TCA precipitate before enzyme inactivation, but that most of the radioisotope became TCA-soluble during enzymatic

TABLE II

Znorganic Phosphate Formation during Enz?]matic Inactivation of LP

To 15.5 mg. of LP in 1.3 ml. was added 0.05 ml. of IE (20 y of protein) and the mixture was incubated at 25”, as were the samples of IE and LP alone. At the indi- cated times small aliquots were removed for activity determinations in the standard LP assay containing B-AMP. Release of inorganic phosphate from LP was meas- ured after sampling 0.2 ml. aliquots into 0.4 ml. of 7.5 per cent TCA. Following centrifugation, 0.4 ml. of the supernatant solution was used in the analysis.

Time

min.

0

50 95

300

I IE LP IE + LP

Y p*

0.03

0.01

7 P per mg. LP Per cent activity

0.01 100

0.02 105 94

y P per mg. LP Per cent activity

0.02 100 0.33 36 0.32 27 0.32 25

* In this case the amount of P found in the TCA supernatant fluid was not related to the mg. of LP but to a volume equivalent to that containing 1 mg. of LP.

inactivation. These results and others (12) indicate that only the phos- phate which is released from LP by IE equilibrates rapidly with phosphate within the slice.

SpeciJidity Studies-The liberation of phosphate from LP prompted a number of experiments to determine whether or not the purified heat- treated inactivating enzyme catalyzed the release of phosphate from other phosphoproteins and non-protein phosphate esters. Non-protein phosphate esters at a final concentration of lo-3 M were incubated in the usual test system, except that caffeine was omitted, since it was not a necessary component for the release of phosphate from LP. 100 to 200 times the usual amount of IE was used and incubations were carried out for as long as 4 hours at 25”. In each case the effect of Mg++ (lO+ M)

was tested. After incubation, 0.5 ml. aliquots were added to 0.8 ml.

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W. D. WOSILAIT AND E. W. SUTHERLAND 477

portions of 10 per cent TCA. Following centrifugation, 1.0 ml. aliquots were analyzed for phosphate by the method of Fiske and Subbarow, adapted to a 1 ml. system. IE was found to liberate no significant amounts of phosphate from a-glycerophosphate, p-nitrophenyl phosphate, 5-AMP, adenosine diphosphate, adenosine triphosphate, creatine phosphate, phos- phoenolpyruvate, glucose-l-phosphate, fructose-l ,6-diphosphate, ribose- 5-phosphate, or pyrophosphate even in the presence of Mg++.

Several known phosphoproteins were incubated with IE under condi- tions similar to those described above. Phosphate was found to be re- leased from phosvitin, and on longer incubation approximately 10 per

TABLE III

Formation of TCA-Soluble Phosphate Measured by Isotopic and Chemical Methods

To 9.5 mg. of LP in 1.9 ml., having a total of 895 c.p.m. (94 c.p.m. per mg. of LP), was added 0.05 ml. of IE (46 y of protein). Incubation was carried out at 25” and 0.5 ml. aliquots were transferred to TCA at the indicated times. After measuring P chemically in the supernatant solution, aliquots of the phosphomolybdate complex were dried and counted. The TCA precipitates were transferred to planchets, dried, and counted.

Incubation time with IE I TCA fraction

min.

0

85

Supernatant Ppt. Supernatant Ppt.

C.p.m. per mg. LP

5 82 75 26*

P per mg. LP

Y

0.13

0.45

A........................................................... 0.32

* Since the precipitates were not washed, an appreciable portion of supernatant solution was present in the precipitate; after correction this value would be lower.

cent of the total phosphate appeared as inorganic phosphate. Smaller amounts of inorganic phosphate were released when cu-casein was the substrate, while no measurable amounts of phosphate were released from p-casein or ovalbumin. A comparison was made of t.he action of IE on LP and phosvitin and, as shown in Table IV, the action of the enzyme on both substrates was inhibited by NaF and NaCl. Enzyme activity was not stimulated by Mg++, and the enzymatic activities were parallel when heat-treated as indicated.

No proteolytic activity was found when purified IE was tested with several proteins as possible substrates.

Characteristics of Inactive LP-The effect of enzymatic inactivation on the sedimentation constant was determined by ultracentrifugal analysis of LP with and without enzymatic inactivation. Prior to inactivation the

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478 INACTIVATION OF LIVER PHOSPHORYLASE

TABLE IV Comparison of IE Activity on LP and Phosvitin

The activity towards LP was studied in the standard IE assay with the indicated modifications. The heated samples of IE were lyophilized samples put through an alcohol step, adjusted with acetate buffer or Tris, heated as indicated, then chilled, neutralized, and tested. The activity towards phosvitin was studied by use of the modified assay in which caffeine was omitted and LP was replaced by 1.8 mg. of di- alyzed phosvitin. The reactions were stopped with TCA after 3 hours incubation at 25” and phosphate was measured in the supernatant fluid.

Treatment

0.15 M NaCl in reaction mixture 0.01 “ NaF “ “ ‘I

0.002 M MgClz in reaction mix- ture

Pretreated for 15 min. at pH 4.0 and 37”

Pretreated for 15 min. at pH 8.0 and 50”

Effect on activity towards

LP I

Phosvitin

7801, inhibition 78yo inhibition 95% “ 70% “ No effect No effect

Complete inactivation Complete inactivation

60-85% ‘recovery 60-85’% recovery

FIG. 2. Ultracentrifugation of LP and inactive LP. Typical sedimentation pat- terns of active LP (upper pattern) and inactive LP (lower pattern) made simultane- ously at 196,900 X g. Two time periods, showing sedimentation at 32 and 40 minutes, are presented to illustrate the character of the peaks observed during sedimentation.

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W. D. WOSILAIT AND E. W. SUTHERLAND 479

samples were dialyzed overnight versus 0.1 M NaCl and 0.002 M Tris, pH 7.4. To 16 mg. of LP in 2.0 ml. of the indicated media was added 0.46 mg. of IE in 0.2 ml. of the same media. The reaction mixture along with the corresponding control (containing all components except IE) was incubated for 1 hour at 25”. During this period the control lost no activity, while in the sample containing IE there was little or no LP activity. Both samples were redialyzed with continual shaking for 2 hours before ultracentrifugation. The samples were centrifuged in a Spinco model D ultracentrifuge at 196,900 X g and at 26.4’2 As indicated in Fig. 2, there was a single symmetrical peak for LP and inactive LP and both had the same sedimentation constant (sZo = 8.4) at identical enzyme concen- trations.2 The displacement of the peaks is a result of different bubble sizes in the two cells. The activities of the suspended samples after centrifugation were essentially the same as before centrifugation. Assay of the reaction mixture in the absence of 5-AMP indicated that 98 per cent inactivation had occurred, while assay in the presence of 5-AMP indicated that 78 per cent inactivation had occurred. As mentioned previously, inactive LP differs from a “b” form of phosphorylase in that enzymatic activity is not restored on addition of 5-AMP to the reaction mixture. Usually when complete inactivation occurred, as judged by assay in the absence of 5-AMP, approximately 85 per cent inactivation occurred, as estimated by assay in the presence of 5-AMP.

DISCUSSION

The study of an enzyme exerting its catalytic action on a substrate in vitro, which is also an intracellular enzyme from the same tissue, posed some special problems. In addition to the problem of obtaining substrate quantities of purified enzyme, the preparation of the substrate (LP) was complicated by the very presence of the catalyst (IE). Moreover, since the substrate was of high molecular weight, it would be expected that the yield of any small fragment would be low in relation to the total amount of substrate used. Experiments reported in this paper showed that the amount of small product formed (inorganic phosphate) was approximately 0.03 per cent of the substrate by weight.

In the standard IE assay the LP concentration was estimated to be 7 X lo-8 M. In liver cells, however, the concentration of phosphorylase is much higher and was estimated to be 1.8 X 10e6 M (a concentration approximately one-tenth that used in the large scale inactivations). The molar concentration of IE present in liver cells cannot yet be estimated,

2 Measurements performed at the Cleveland Clinic. M. D. Schoenberg, personal communication.

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480 INACTIVATION OF LIVER PHOSPHORYLASE

but the enzyme concentration in liver is sufficient to catalyze the inactiva- tion of all LP present in several minutes or less.

All evidence to date indicates that IE is a phosphatase. No materials except phosphate and inactive LP were found to be products of the in- activation reaction; hence liver phosphorylase phosphatase might be considered an appropriate name for the enzyme. Evidence supporting this view is reported in Paper III (12) where it is shown that inactive LP, after extensive dialysis, could be reactivated in the presence of another enzyme fraction and adenosine triphosphate. Phosphatase activity was not exhibited when IE was incubated with a number of simple organic phosphate esters, but some phosphate was released from phosvitin and cY-casein. The amounts of phosphate released were small, however, and it is not entirely clear that phosphoserine proteins are the necessary sub- strates for IE. The nature of the organic phosphate moiety in LP is currently under investigation and future results may permit a more exact descriptive name for the enzyme.

The dephosphophosphorylase (inactive LP) formed during the reaction sedimented at the same rate as the active form. Thus, there did not appear to be a gross change in the phosphorylase molecule, as was re- ported when phosphorylase a from rabbit muscle was incubated with the phosphorylase-rupturing enzyme (13). Therefore, the exact relationship of IE to the phosphorylase-rupturing enzyme is not clear, especially since the enzymes studied were different in origin and properties.

Results to be published at a later date indicate that IE from dog heart muscle is similar to the one from dog liver. Highly purified inactivating enzyme from dog heart was also found to catalyze the release of inorganic phosphate from liver phosphorylase as did the IE from dog liver.

SUMMARY

1. The purification and properties of a soluble liver phosphorylase- inactivating enzyme from dog liver have been described.

2. The inactivating enzyme catalyzed the conversion of liver phosphor- ylase to an inactive form whose activity was not restored by adenosine-5- phosphate. Products of the enzymatic inactivation were inactive liver phosphorylase and inorganic phosphate. Inactive liver phosphorylase sedimented at the same rate as the active form.

3. The inactivating enzyme exhibited phosphatase action when incu- bated with liver phosphorylase and slight phosphatase activity was noted on incubation with phosvitin and a+casein. No phosphatase activity was noted, however, on incubation with a number of simple organic phosphate esters.

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W. D. WOSILAIT AND E. W. SUTHERLAND 481

The authors wish to thank Miss Arleen Maxwell and Mr. James Davis for technical assistance in these studies.

BIBLIOGRAPHY

1. Sutherland, E. W., Ann. New York Aced. Xc., 64,693 (1951). 2. Sutherland, E. W., and Wosilait, W. D., Nature, 176, 169 (1955). 3. Wosilait, W. D., Federation Proc., 14, 307 (1955). 4. Sutherland, E. W., and Wosilait, W. D., .I. Biol. Chem., 218, 459 (1956). 5. Fiske, C. H., and Subbarow, Y., J. Biol. Che*rr., 66, 375 (1925). 6. Lowry, 0. H., and Lopez, J. A., J. Biol. Chem., 162, 421 (1946). 7. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

tissue metabolism, Minneapolis, 191 (1949). 8. Seifter, S., Dayton, S., Novic, B., and Muntwyler, E., Arch. Rio&em., 26, 191

(1950). 9. Nelson, N., J. Biol. &em., 163, 375 (1944).

10. Bessey, 0. A., Lowry, 0. H., and Brock, M. J., J. Biol. Chem., 168, 197 (1947). 11. Weichselbaum, T. E., Am. J. Clin. Path., 16, Tech. Suppl., lo,40 (1946). 12. Rall, T. W., Sutherland, E. W., and Wosilait, W. D., J. BioZ. Chem., 218, 483

(1956). 13. Keller, P. J., and Cori, G. T., J. Biol. Chem., 214, 127 (1955).

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Walter D. Wosilait and Earl W. SutherlandPHOSPHORYLASE

INACTIVATION OF LIVER PHOSPHORYLASE: II. ENZYMATIC

AND GLUCAGON TO LIVER THE RELATIONSHIP OF EPINEPHRINE

1956, 218:469-481.J. Biol. Chem. 

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