Endocrinol. Japon 1959, 6 (3), 191～200

STUDIES ON UROPAROTIN

IV. ISOLATION AND SOME PROPERTIES OF UROPAROTIN

YOSOJI ITO

Department of Physiological Chemistry,Faculty of Pharmaceutical Sciences,

University of Tokyo

MASAAKI YAMAMOTO

Research Laboratory, Teikoku Hormone Mfg. Co., Ltd.,Kawasaki

In the previous paper, it was reported that a substance having similar

biological effects to salivary gland hormone(parotin)and termed as uroparotin

(Ito et al., 1954)was detected in human urine. The extraction and purificationof uroparotin have been carried out and a protein-like substance was given(Ito

and Yamamoto, 1958a, b). Furthermore, a periodical seasonal variation in itsbiological activity was confirmed, and it was found that the active type of uro-

parotin(ƒ¿-Urp)was excreted only in summer and the inactive type(ƒÀ-Urp)in

winter(Ito and Yamamoto, 1959).

According to the results of previous experiments, ƒÀ-uroparotin is already an

inactive form when it is excreted in urine being easily converted to a-uroparotin

by incubation at 37•Ž(Ito and Yamamoto, 1959). These facts led the authors

to assume some differences in the occurrences of activity of a-and(3-uroparotin

due to different chemical or physical properties. Isolation of both uroparotins in

a homogeneous state was therefore attempted in order to elucidate the mecha-

nism of occurrence of the biological potencies by means of comparing chemical

properties of both isolated uroparotins.

EXPERIMENTAL

Isolation procedure of uroparotinBoth a-and ƒÀ-uroparotins were isolated with the exactly same procedure. The original

urine was collected from healthy young men by adding toluene, during summer from June to

September for a-uroparotin and during winter from December to March for ƒÀ-uroparotin. Each

step of the isolation procedure was as follows;

Step 1(Extraction of crude uroparotin)Crude uroparotin was obtained from human urine by means of the acetone precipitation method

followed by the isoelectric concentration method described in the previous paper(Ito and

Ya:namoto, 1958a). The obtained crude uroparotin was termed as"FR"and used as thestarting material for the succeeding purifications.

Received for publication July 6, 1959.

192 ITO AND YAMA MOTO Vol. 6, No. 3

Step 2(Ethanol fractionation)Crude uroparotin(FR)was dissolved in 0.2M sodium chloride solution to make 1.5% con-

centration adjusting to pH7.0.To the clear supernatant obtained after centrifugation,ethanol

was added by stirring to make a 40% concentration at 0•Ž and a small amount of precipitate

was discarded by centrifugation. Ethanol was further added to the supernatant with stirring to

make a 60% concentration at 0•Ž and the solution was allowed to stand overnight in an ice-

room before centrifugation to remove the precipitate. The obtained precipitate was dissolved

in a small amount of water and dialyzed against distilled water. This ethanol fractionation was

repeated again for the dialysate. The final precipitate from 60% ethanol was dissolved in a

small amount of water, dialyzed against distilled water, and lyophilized.This dry powder was

termed as "FE".

Step 3(Alumina column treatment)

FE was further purified by the alumina column method. The previously reported method(Ito

and Yamamoto, 1958b)was modified to be more convenient for alumina column chromatography.

The reproducibility of this procedure was satisfactory and a homogeneous uroparotin was

obtained with a eood vield.

Previously, alumina was washed with distilled water more than several times to remove the

fines with the decantation. The washing was carried out repeatedly until the pH of the sus-

pension was brought under 7.5. The washing with hydrochloric acid should be avoided owing

to the effect increasing the anion exchange effect in alumina and also the unsatisfactory repro-

ducibility.

1st alumina column treatment:Alumina suspended in distilled water was tightly filled into a

column of 2.5•~40cm and was washed thoroughly with distilled water. Five hundred mg of

FE was dissolved in water to make a 5% concentration and applied at the upper site of the

column before elution with distilled water. The effluents were collected in the tubes every 10

ml with the fraction collector and optical density at 277mƒÊ was determined in each fractions.

The effluents in the tubes from 9th to 20th showing an adsorption peak were mixed,and after sodium

chloride was added to make 0.2M, 4 volumes of ethanol was added with stirring to make a

80% concentration. The precipitate thus obtained was separated by centrifugation and dissolved

in a small amount of water, dialyzed against water, and lyophilized. This powder was named

as FC1.

2nd alumina column treatment: Alumina was filled into a column of a size of 2.0•~30cm and

washed with water.Then,150mg of FC1 was applied to the column and eluted with distilled

water.The effluents were collected every 10ml and the tubes from 7th to 15th showing an

adsorption peak at 277mƒÊ were mixed and centrifuged. From the clear supernatant, a pre-

cipitate was obtained by the same ethanol concentration method. It was dissolved in small

amount of water and dialized and lyophilized. The resulted powder was termed as FC2.

Final alumina treatment: Alumina was filled into a column of a size of 1.2×30cm and washed

thoroughly with water. Eighty mg of FC2 was dissolved in water to make a 5% concentration,

applied to the column, washed, and eluted with distilled water. Among the effluents collected

in the tubes every 5ml with the fraction collector,the tubes from 5th to 12th showing an

adsorption peak at 277mƒÊ were mixed together. After centrifugation, the precipitate was

obtained with the same ethanol concentration method from the clear supernatant. The preci-

pitate was dissolved in a small amount of water,dialyzed against distilled water, and lyophilized.

The finally obtained powder was termed as FC3 and designated uroparotin.

Bioassay

The biological activity was estimated by serum-Ca reduction method and circulating leu-

cocyte reaction method which have already been reported in the previous paper(Ito and Yama-

moto, 1958a). Each activity was termed as Ca-activity and as L-activity, respectively.

Homogeneity of uroparotin

The homogeneity of uroparotin was tested with both electrophoresis and ultracentrifugation.

Sept.1959 STUDIES ON UROPA ROTIN 193

For electrophoresis, the following three buffers were used; pH 8.6, ƒÊ=0.1, veronal-sodium

veronal, pH 5.1, ƒÊ=0.1, acetic acid-sodium acetate and pH2.0, ƒÊ=0.2, glycine-hydrochloric

acid. And the patterns were obtained after the moving at 8mA for 45mins. to 60mins.

Ultracentrifuge analysis was carried out under the condition of 57,800 r.p.m. using pH7.8

phosphate buffer.

Amino acid composition

For the analysis of amino acid composition of uroparotin, 3 to 5mg of uroparotin were

hydrolized in a sealed tube with 0.5ml of constant boiling hydrochloric acid for 24 hrs. at 110•Ž

and with 1ml of 0.5 N Ba(OH)9 for 24hrs. at 125•Ž. The hydrolysates were applied on a

Whatman No. 1 filter paper for analysis by two dimensional chromatography in the solvent sys-

tem of n-butanol-acetic acid-water(4: 1: 2), followed by phenol saturated with 0.3% ammonia

for acid hydrolysate, or followed by n-butanol saturated water for alkaline hydrolysate. The

paper was sprayed with a solution of ninhydrin to produce the chromatogram. Three parts in

which the spots could not be readily distinguished, namely basic amino acid group of lysine,

arginine and histidine, the group containing valine and methionine, and the group containing leucine,

isoleucine and phenylalanine, were cut out. After the extraction with 0.1 N ammonia, each extract

was applied on a circular filter paper(diameter of 30 cm) for circular chromatography in the solvents

of s-collidine-2.6-lutidine-water(1: 1: 1) added diethylamine (Dent, 1947), pyridine-acetic acid-

water(50: 35: 15) (Decker and Riffart, 1950), and developed along with the reference amino

acids. The contents of tyrosine and tryptophan were estimated from the ultraviolet light absorp-

tion of intact uroparotin according to the procedure of Goodwin and Morton(1946).

Estimation of isoelectric point

Although the measurement of isoelectric point using paper electrophoresis has been reported

in Paper 2 of this series, it was not possible to obtain an accurate result in this experiment due

to the high salt concentration of the buffers. In the present experiment, the influence of electro-

osmotic flow due to filter paper was not only corrected with non-charged dextran, but also

bovine serum albumin was used together as the reference protein according to the procedure of

Raacke and Li(1955). Four sample solutions of a-uroparotin, ƒÀ-uroparotin, bovine serum albumin

and dextran were applied on a Whatman No. 1 filter paper and moved together under the con-

dition of various pH and current of 3mA/cm, for 6 hrs. A detail of the procedure was the

same as in the previous paper.

Color reaction of uroparotin

In order to clarify the chemical characteristics of uroparotin, the following color reactions were

carried out qualitatively; Ninhydrin, Biuret and Millon reaction for protein analysis, Molisch and

Orcinol reaction for carbohydrate analysis, especially Bial's, direct Ehrlich and Diphenylamine

reaction(Werner and Odin, 1952) for sialic acid, Sudan black B reaction for lipoprotein, and

color reaction of phosphorous after a hydrolysis using perchloric acid for phosphoprotein.

Estimation of proteolytic activities

Proteolytic activities of uroparotin were studied with the tyrosine ultraviolet light absorp-

tion method described by Kunitz(1947) and casein was used as the substrate. For the estima-

tion of peptic action, uroparotin has been incubated previously with hydrochloric acid and then

reacted with the casein solution at pH1.6 at 37•Ž for 10mins. For tryptic action, intact

uroparotin was reacted with the casein solution at pH 7.8 at 37•Ž for 10mins. The proteolytic

activities were expressed in terms of r tyrosine released in a minute.

The effects of uroparotin on the blood pressure

In order to confirm whether oxytocic or callicrein effect was detected in uroparotin, the

blood pressure depressing effect was estimated using a chicken and a dog, respectively.

Other hormonal action of uroparotin

Corticotrophic action of uroparotin was estimated by means of Sayer's method using Scher-

ing-ACTH as the standard preparation. Gonadotropic action was tested by the rat ovary weight

method and the pigeon method was used for the estimation of lactogenic action.

194 ITO AND YAMAMOTO Vol. 6, No. 3

RESULTS

The finally purified product(FC3) prepared through the above mentioned

steps was designated as uroparotin. The yields and estimated activities of all

fractions are shown in Table 1. It might be noted that 1-1.2mg of ƒ¿-uroparotin

and 0.8mg of A-uroparotin were obtained from 1 liter of human urine. The

lower yield of ƒÀ-uroparotin seemed to be due to the lower concentration of

uroparotin in winter urine.

Table 1. Yield and activity of various fractions obtained

from procedure for isolation of uroparotin

Fig. 1. The purification of uroparotin

by the alumina column treatment method.

(Upper part): 500mg of FE was appli-

ed on 2.5•~40cm column.

Eluents: (1) distilled

water,(11) 0.1 M pH

6.90 phosphate buffer

(Middle part): 150mg of FC1 was ap-

plied on 2.0•~30cm

column. Eluents:(I) dis-

tilled water,(II) 0.1 M

pH6.90 phosphate buffer

(Lower part): 80mg of FC2 was ap-

plied on 1.2•~30cm

column. Eluents:(I) dis-

tilled water,(II) 0.1 M pH

6.90 phosphate buffer

Scpt. 1959 STUDIES ON UROPAROTIN 195

(A)

(B)

(C)

Ascend Descend

Fig. 2. Electrophoretic pat-

terns of uroparotin

(A) pH 8.6, ƒÊ=0.1, veronal

buffer, 8 mA, 45 mins.

(B) pH5.1, ƒÊ=0.1, acetic

acid buffer, 8mA, 45mins.

(C) pH2.0, ƒÊ=0.2, glycine

buffer, 8mA, 60mins.

Fig. 3. Ultracentri-

fuge of uroparotin

pH7.8 phosphatebuffer, 59,789 r. p. m.

45mins.

Although the alumina column treatment was merely a repetition of the same

technique with various scales of column, its purifying effect was sufficientlysatisfactory as shown in Figure 1; in the first column treatment, an active frac-

tion(FC1) was eluted with distilled water. Then, by elution with 0.1 M Na-phos-

phate buffer, band(Al) having a little activity was obtained and after alterationof pH, two inactive bands(B1, C,) were eluted. In the second column treatment,

α、uroporotin

β、uroparotin

bovine serum albumjn

Fig. 4. Migration of a-, ƒÀ-uroparotin and bovine serum

albumin under various pH

196 ITO AND YAMAMOTO Vol. 6, No. 3

(u-A)

(u-B)

(u-C)

Phenol(0.3% NH3)

Fig. 5. Two dimensional paper chromatogramof uroparotin hydrolysate with hydrochloric acid1. Cys, 2. Asp, 3. Glu, 4. Ser, 5. Gly, 6. Thr, 7.Ala, 8. Tyr, 9. Lys, 10. His, 11, Arg, 12. Met(Val), 13. Leu(lieu), 14. Phe, 15. Pro

FC1 was applied to the column

and an active fraction FC2 was

eluted with distilled water, thenA2 and B2 were obtained with

phosphate buffer. The lattertwo bands were smaller than

those of Al and B1, respectively,especially the yield from the

elution band B2 after alteration

of pH was extremely low. Inthe final treatment, FC2 was

applied to the column. An ac-tive fraction FC3 was concen-

trated by elution with distilled

water and only a trace of elu-tion band(A3) followed it with

phosphate buffer. As the result,final fraction FC3 was given in

a homogeneous form chromato-

graphically.

The homogeneity of uroparotin was detected by both methods of electro-

phoresis and ultracentrifugation, and ƒ¿-and ƒÀ-uroparotins were exactly identical.

Hereby, the result with a-uroparotin is shown in Figures 2-3; with electrophoretic

patterns in three different pH(8.6, 5.1 and 2.0) and with ultracentrifuge pattern

in pH7.8 phosphate buffer, it was confirmed that uroparotin is a homogeneous

component.

To estimate the isoelectric point, paper electrophoresis was carried out under

the condition of various pH. The distances of migration of a-, ƒÀ-uroparotin and

bovine serum albumin were corrected by dextran and plotted against pH as

shown in Figure 4; the pH-distance curves of a-and ƒÀ-uroparotin were exactly

in accordance with each othershowing that the isoelectric pointswere equal and were approxi-mately parallel to the curve ofbovine serum albumin. If theisoelectric point of bovine serumalbumin is considered to be pH4.6, that of a-and j3-uroparotinmay be estimated from the presentresult, which is shown in Figure4, as follows;

2.0+(4.6-3.7)=2.9

The results of two dimensio-

nal paper chromatogram for the

qualitative analysis of amino acidsare shown in Figures 5-6. Only

n-Butanoi+acetic acid +water(4: 1: 2)

Fig. 6. Two dimensional paper chromato-

gram of uroparotin hydrolysate with baryta1. Pro, 2. Tyr, 3. Try, 4. Met, P. Peptide

Sept.1959 STUDIES ON UROPAROTIN 197

Fig. 7. Circular paper chromatogram of uroparotin hydrolysate.The sample solutions from U-A, U-B, and U-C were applied on What-man No. 1 filter paper. Developed with solvent of(left part) s-collidine-2.6-lutidine-water(1:1:1) added diethylamine and(right part)pyridine-acetic acid-water(50:35:15). Sprayed with ninhydrin reagent.Referenced amino acids were as follows;1. Phenylalanine(greenish blue), 2. Leucine+isoleucine(purple),3. Methionine(brown), 4. Valine(purple), 5. Histidine(purple),6. Arginine(greenish blue), 7. Lysine(purple), 8. Phenylalanine+leucine(purple), 9. Isoleucine(blue-purple)-

the result with a-uroparotin was pre-

sented for the similarity of results ob-

tained with both types of uroparotin.

All three parts(U-A, U-B, U-C), in

which the separation of each component

was insufficient; containing leucine+iso-

leucine+phenylalanine, valine+methio-

nine, lysine+histidine+arginine, respec-

tively, were cut out, and then circular

paper chromatography was applied inorder to identify amino acids. The

result is shown in Figure 7. Among

these amino acids, valine could not be

detected. It will be seen that the com-

monly occurring 17 amino acids are pre-

sent in uroparotin, only except valine;

cystine, aspartic acid, glutamic acid,

serine, glycine, threonine, alanine, tyro-

sine, methionine, proline, phenylalanine,

leucine, isoleucine, lysine, histidine, argi-

nine and tryptophan. The effects on

blood pressure of uroparotin are shown

in Figure 8, A and B; uroparotin had no

falling effect on blood pressure in chicken

and dog.

All properties of uroparotin obtained

in the experiment are summarized in

(A)

(B)

Fig. 8. Effect of uroparotin on blood

pressure of dog and chicken

A.(dog) S: Callicrein standard 0.15u.

U1: a-uroparotin 0,6mg

U2: •V •V If 2.1mg

B.(chicken) S: Oxytocin standard 0.02 i.u.

Urpl: a-uroparotin 0.6mg

Urp2: •V •V 1.5mg

After injection of uroparotin, a slightly ris-

ing effect on blood pressure appeared.

198 ITO AND YAMA MOTO Vol. 6, No. 3

Table 2. Properties of uroparotin(1)

Tables 2 and 3. The property of glycoprotein was observed with uroparotin andespecially, it was noticed that sialic acid might exist a carbohydrate moiety.Proteolytic activities were hard to detect since an activity of 1/400 or less thanthe crystal line proteiase was estimated. The contamination of other hormonalsubstances in uroparotin could not be observed completely.

Table 3. Properties of uroparotin(2)Proteolytic and other hormonal activities

*: Crystalline pepsin [PU]=

Cas 275

900/mgγ,tyr

Crystalline trypsin [pu]=

Cas 275

1500/mgγ,tyr

Sept. 1959 STUDIES ON UROPA ROTIN 199

DISCUSSION

The potency of an active uroparotin(ƒ¿-uroparotin)is revealed through the

minimum effective dose of 0.1mg/kg, being far more active than"parotin"iso-

lated from bovine salivary gland. The studies concerning the relationship between

uroparotin and salivary gland are being carried out using such experimental

animals as rabbit and dog, etc. As for salivary gland hormone of human subject,

no study has been reported so far. Therefore, at the present stage, it is hardly

significant to compare the activity of uroparotin with other salivary gland hor-

mones(parotin, saliva-parotin, and S-parotin).

Uroparotin is considered to be a glycoprotein from its color reactions and it

is interesting that sialic acid is detected as one component of carbohydrate

moieties.

Previously, Weiner et at.(1950), and Schmid(1953, 1955)have reported the

isolation of glycoprotein containing sialic acid from serum, and it has been pointed

out that the low isoelectric point of their substance is due to the contained sialic

acid(Odin and Werner, 1952, Schmid, 1959).

In view of this fact, a similarity between uroparotin and serum glycoprotein

is suggested with regard to the low isoelectric point. It is of interest,therefore,

to prepare acid-glycoprotein from serum according Schmid to test whether the

material has any uroparotin-like biological activity.

However, callicrein has been reported to appear in urine(Frey and Kraut,

1926)and possesses a depressing effect on blood pressure. Uroparotin is not

identical with callicrein because of the absence of effects on a phenobarbital-

anesthetized dog. Similarly, the oxytocic and other hormonal actions were not

observed in uroparotin, so that the contamination with these hormone-like sub-

stances was ruled out. The descriptions that pepsinogen(uropesin)appears in

human urine(Hirschowitz, 1957)and its excretion is influenced by seasonal varia-

tion(Sakurai. 1958), have been also reported, and the seasonal variation was

similar to that of uroparotin.Proteolytic activity of uroparotin was therefore

estimated but no activity was found.

Uroparotin is a noticeable substance as glycoprotein appearing in urine owing

to the characteristic and the seasonal variation in biological potencies.Although

both active and inactive types were observed in uroparotin, no difference in the

properties of both types could be found from the present studies, and all pro-

perties were identical to each other.Consequently, studies concerning the

mechanism of introducing the biological effect of uroparotin must be carried out

in the following stage.

SUMMARY

By means of isoelectric precipitation, ethanol fractionation,and alumina

treatment method, uroparotin was isolated in a homogeneous form from human

urine. The yield of uroparotin was shown to be 1-1.2mg per liter for a-uroparo-

tin and 0.8mg per liter ƒÀ-uroparotin. Uroparotin was considered to be a typical

glycoprotein, containing sialic acid as one component of carbohydrate moieties.

200 ITO AND YAMA MOTO Vol. 6, No. 3

Neither a proteolytic effect nor a blood pressure depressing effect was observed

in uroparotin, nor other hormonal actions. Therefore, uroparotin may be con-

sidered a new glycoprotein having characteristic biological effects.No difference

in properties of both a-and ƒÀ-uroparotin could be found,and the mechanism of

the biological effects remains to be clarified.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to Miss Yoshiko Ishida for hercooperation during the course of this study. The authors are also grateful to the

staffs of the department of biological research, Teikoku Hormone Mfg. Co., Ltd.,for their technical assistances on bioassay of uroparotin.

REFERENCES

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