Studies on phosphorylase isozymes in lower vertebrates: Evidence for the presence of two isozymes in elasmobranchs

ARCHIVES Vol. 197,

OF BIOCHEMISTRY AND BIOPHYSICS No. 1, October 1, pp. 149-157, 1979

Studies

on Phosphorylase lsozymes in Lower Vertebrates: Evidence for the Presence of Two lsozymes in Elasmobranchsl SATOSHI Department

YONEZAWA of Zoology,

Received

AND

Hokkaido

January

SAMUEL

University,

8, 1979; revised

H. HORI

Sapporo,

060 Japan

June 6, 1979

Two distinct phosphorylase isozymes, skeletal muscle phosphorylase b and liver phosphorylase 6, have been purified from skate (Raja pulchra) in a homogeneous form as judged by electrophoretic and immunological criteria. Both isozymes were dependent on AMP for activity and converted to a forms by rabbit muscle phosphorylase kinase. Their subunit molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was 94,000. These isozymes were distinctly different in affinities for glycogen and AMP, while they were very similar in sensitivities to SO:-. Rabbit antibodies against each of the muscle and liver isozymes inhibited completely the respective specific antigens. No cross-reaction was observed in double diffusion tests, but some immunological relatedness of these isozymes was demonstrated by inhibition tests with antibodies. Their similarity was also shown by amino acid analyses. No evidence has been obtained that the skate possesses such an isozyme as mammalian phosphorylase L, the b form of which is inactive even in the presence of AMP. Electrophoretic studies on phosphorylases of crucian carp, toad, and snake revealed that these animals possess three isozymes which strikingly resemble mammalian isozymes in the organ-specific distribution and electrophoretic behavior.

Evidence has been presented that mammalian species possess three isozymes of phosphorylase (EC 2.4.1.1) (l-6). They are very similar with respect to the interconversion of the active (phospho, a) and inactive (dephospho, b) forms (2, 5, 7-9), amino acid composition (g-11), and subunit molecular weight (2, 8, 9, 12). Formation of hybrid molecules has also been noted between these isozymes (2, 3, 6, 7). Such similarities strongly suggest that the isozymes have diverged from a common ancestral molecule, but we have as yet little knowledge about evolutionary aspects of phosphorylase isozymes. In a previous paper (13), we showed that lamprey, one of the most primitive vertebrates, possesses only one molecular form of phosphorylase. When compared with mammalian isozymes2 lamprey phos1 This is part II in a series. For part I see Ref. (13). ’ The nomenclature for mammalian phosphorylase isozymes used in this paper was described previously (6).

phorylase not only resembles mammalian isozymes III and 1 with respect to the AMPdependence of the b form, but also resembles isozyme L with respect to the insensitivity to mercaptans and the activation by SO:-. Accordingly, it seems highly probable that early in the evolution of vertebrates there was only a single phosphorylase gene, and that this ancestral gene underwent gene duplication at least twice to yield more differentiated forms of phosphorylase during vertebrate evolution. In this connection, our previous findings are of interest, in that relative inhibition potencies of antilamprey phosphorylase antibody are significantly different with skeletal muscle phosphorylase and with liver phosphorylase of the skate and shark (13). This strongly suggests that elasmobranchs possess at least two phosphorylase isozymes. The purpose of the present study is to characterize these isozymes and to discuss their physiological significance. A preliminary observation that crucian carps, toads, and snakes 149

0003-9861/79/110149-09$02.00/O Copyright All righls

0 1979

by Acaclemic

of reproduction

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Inc.

in any form reserved.

150

YONEZAWA

possess three phosphorylase isozymes which resemble the three mammalian isozymes in organ-specific distribution and electrophoretic behavior will also be described for the sake of comparison with elasmobranch isozymes. MATERIALS

AND METHODS

Enzyme assays, kinetic studies, and purification of lamprey phosphorylase b and rabbit muscle phosphorylase b kinase were carried out by the methods reported previously (6, 13). Rabbit muscle phosphorylase b was purified according to Fischer and Krebs (14). Protein was determined according to Lowry et al. (15), using bovine serum albumin as a standard. Electrophoretic studies. Polyacrylamide gel electrophoresis of phosphorylase was carried out as described previously (6). Tissue extracts were prepared in the presence or absence of 0.1 M NaF (6) from tissues of skate (Raj, pulchra), shark (Squab acanthias), crucian carp (Carassius carassius), rainbow trout (Salmo gairdneri irideus), toad (Bufo bufo), and snake (Elaphe climacophora). The estimation of molecular weight was carried out by the methods of Weber and Osborn (16) and of Zwaan (1’7). Immunological studies. Antisera against skate phosphorylase isozymes were prepared in rabbits according to the procedure described previously (13). Antigen doses were 1 mg per injection in the case of the muscle enzyme and 0.5 mg per injection in the case of the liver enzyme. For determining relative inhibition potencies of antibodies (4, 5), antisera were treated with (NH&SO+ and the precipitate at 33% saturation was used as antibody after dialyzing against 0.9% NaCl. Heterologous phosphorylases were partially purified in the b form by (NH&SO4 fractionation in the same way as for skate muscle phosphorylase (see Results), except that crucian carp liver phosphorylase a was isolated through DEAEcellulose chromatography according to Wolf et al. (9). Ouchterlony agar diffusion tests were carried out as described previously (13). Amino acid analysis. The amino acid compositions were analyzed by the procedure of Moore and Stein (18) using a JEOL 6AH or a Hitachi 835 automatic amino acid analyzer. Hydrolysis was performed with 6 N HCl at 107 + 1°C for 16, 24, 48, and 72 h. The values for threonine and serine were extrapolated to zero time. Tryptophan was determined spectrophotometrically by the procedure of Bencze and Schmid (19). The total cysteine content was determined as cysteic acid after oxidation with performic acid according to Moore (20) or as free sulfhydryl groups in the sodium dodecyl sulfate (SDS)3-dena3 Abbreviations used: SDS, Sodium dodecyl sulfate; RIP, relative inhibition potency.

AND HORI tured protein measured by titration dithiobis(2-nitrobenzoic acid) (21).

with

5,5’-

RESULTS

Organ Speci$city of Phosphorylase Isoxymes in Skate

Figure 1 shows phosphorylase b isozyme patterns of skeletal muscle, heart, and liver in the skate. Single bands of different mobility were found in skeletal muscle and liver, while heart, brain, kidney, spleen, and intestine showed three bands; the slowand fast-moving bands migrated as fast as skeletal muscle and liver phosphorylases, respectively. The intermediate band was identified as the hybrid molecule between the muscle and liver isozymes, because it was readily formed in a mixture of muscle and liver extracts (Fig. Id). Addition of NaF to the homogenizing buffer was without effect on the isozyme patterns. A similar tissue-specific distribution of isozymes was also observed with shark tissues. These electrophoretic findings indicate that elasmobranchs have two isozymes of phosphorylase, one is predominant in the skeletal muscle and the other in the liver, as predicted in a previous report (13). Putification of Skate Muscle Phosphorylase b

Crude extracts from frozen skate muscle, about 100 g, were prepared in the same way as for lamprey muscle phosphorylase (13), and treated with (NH&SO, at 33-50% saturation. The precipitate was dialyzed overnight against 20 IrIM triethanolamine a

b

c

d

FIG. 1. Phosphorylase b isozyme patterns of skate tissues. a, skeletal muscle; b, heart; c, liver; d, the mixture of skeletal muscle and liver extracts. The gel electrophoreses were carried out as described previously (6).

PHOSPHORYLASE

ISOZYMES

IN THE

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buffer, pH 7.5 (1 mM EDTA and ‘7 mM mercaptoethanol were added to all buffers used) with several changes. After centrifuging at 105,OOOg for 40 min, the enzyme solution was applied on a 2.2 x lo-cm DEAE Sephadex A-50 column which had been equilibrated with the same buffer. The column was washed extensively with the buffer, and the enzyme was eluted with a linear gradient formed from 150 ml each of the above buffer and 0.5 M NaCl in the buffer. Flow rate was adjusted to 30 ml/h. Fractions of 5 ml were collected, and those with high enzyme activity were pooled and immediately layered on a 2.2 x 2-cm column of 5’-AMP Sepharose 4B equilibrated previously with the triethanolamine buffer. The column was washed with the buffer at a flow rate of 30 ml/h and the enzyme was eluted with 5 mM AMP in the buffer. A summary of a typical purification of skate muscle phosphorylase b is shown in Table I.

mixed with an equal volume of neutral saturated (NH&SO,. After standing at 4°C for 2 h, the precipitate was collected by centrifugation and dissolved in about 30 ml of 2 mM glycerophosphate buffer, pH 7.2, containing 1.5% skate liver glycogen, which was ‘purified according to the procedure of Somogyi (22). The mixture was then centrifuged at 105,OOOg for 60 min, and the enzyme precipitate with glycogen was dialyzed against 2 mM glycerophosphate buffer, pH 7.2, and layered on a 1.3 x locm column of DEAE-cellulose which had been equilibrated with the same buffer. After washing with the buffer, phosphorylase was eluted with a linear gradient formed from 100 ml each of 2 mM glycerophosphate buffer, pH 7.2 and 100 mM glycerophosphate buffer, pH 6.2, at a flow rate of 20 ml/h, and the eluate was collected in 4-ml fractions. The fractions containing high enzyme activity were treated with (NHJ2S04, and the precipitate obtained between 35 and 50% saturation was dialyzed overnight at 4°C against 20 mM Puri$cation of Skate Liver Phosphorylase b Tris buffer, pH 7.2. After removing inFrozen skate liver, about 200 g, was soluble materials by centrifuging, the soluthawed and homogenized for 30 s with 2 vol tion was applied to a 1 x 5-cm column of Sepharose 4B which had been of distilled water in a Waring Blendor at 5’-AMP with the Tris buffer. The 0°C. The mixture was centrifuged at 8000g equilibrated for 20 min, and the supernatant was care- column was washed with this buffer at a fully decanted. After addition of EDTA and flow rate of 12 ml/h and the enzyme was mercaptoethanol at final concentrations of 1 eluted with 1 mM AMP in the buffer. Fractions of 1 ml were collected and those and 7 mM, respectively, the supernatant was made to pH 5.4 with 1 M acetic acid, with high enzyme activity were pooled. allowed to stand at 4°C for 30 min, and Table II shows a summary of one typical centrifuged at 8OOOgfor 10 min. The resultpurification of skate liver phosphorylase b. ing supernatant was passed through glass The enzyme preparations thus prepared wool, neutralized with solid NaHCO,, and were freed of AMP by passing through a TABLE PURIFICATIONOFSKATE Total volume (ml)

Fraction Crude extract= 33-50% (NH&SO, DEAE Sephadex chromatography Affinity chromatography ’ Starting

material,

MUSCLE Total

activity

(units)

284 40

4824 4600

108 14

3456 3045

110 g of frozen

skate

I

muscle.

PHOSPHORYLASE Total protein (mg) 2542 572 61.7 38.6

b Specific (units/mg

activity of protein)

Recovery (%)

1.9 8.0

100 95

56.0 78.8

72 63

YONEZAWA TABLE

AND

HORI

II

PURIFICATIONOF SKATE LIVER PHOSPHORYLASE b Total volume (ml)

Fraction Crude extracta pH 5.4 supernatant 50% (NH&SO, Glycogen precipitation DEAE-cellulose chromatography 35-50% (NH&SO, Affinity chromatography n Starting

material,

Total activity

375 355 41 18

863 751 613 449

40 5.5 4.0 266 g of frozen

(units)

267 190 129 skate

Sephadex G-25 column equilibrated with 2 glycerophosphate buffer, pH 7.2, and used in the following studies. and Molecular

Weight

The specific activity of muscle phosphorylase b was about 80 and 2 unitslmg of protein in the presence and absence of 1 mM AMP, respectively, while that of liver phosphorylase b was about ‘75 and 2 units/mg of protein, respectively. The specific activities of the a forms which were prepared in vitro from the b forms by the previous method (13) were essentially the same as those of the b forms when assayed in the presence of 1 lllM AMP, while in the absence of AMP, muscle phosphorylase a and liver phosphorylase a had 66 and 65 units of activity/mg of protein, respectively. The muscle and liver enzymes were both electrophoretically pure (Fig. 2). Their molecular weights estimated electrophoretically were about the same, being 94,000 on SDS gels, and 173,000 on SDSfree gels. This suggests that each enzyme exists in a dimeric form of identical subunits. Optimal

pH and Kinetic

Specific (units/mg

5753 3413 1393 54.6 8.37 3.66 1.71

activity of protein) 0.15 0.22 0.44 8.22

31.9 51.9 75.4

Recovery (%I 100 87 71 52 31 22 15

liver.

InM

Purity

Total protein (mg)

for glycogen and AMP than the liver isozyme, while they were similar in respect to the affinity for glucose l-phosphate. When compared with available data (4, 13, 23281, it appears that skate muscle phosphorylase has kinetic properties similar to those of the muscle enzymes from sharks and mammals, while skate liver phosphorylase rather resembles lamprey phosphorylase. Effects of Na,SO, Effects of Na,SO, are illustrated in Fig. 3. In the presence of AMP, the a and b forms of skate phosphorylases were in-

Properties

A broad pH optimum ranging from pH 6.1 to pH 6.8 was observed with the muscle and liver phosphorylases. Table III shows the K, values at pH 6.8 of the a and b forms of the isozymes. The muscle isozyme had higher affinities

FIG. 2. SDS-gel electrophoresis of purified muscle phosphorylase b (20 pg; gel 1) and liver phosphorylase b (33 pg; gel 2). Gels were stained with amido black.

PHOSPHORYLASE TABLE

Glucose l-phosphate (mm

Muscle a

b Liver a

b

III

OFSKATE PHOSPHORYLASES

KmVALuEs

Enzyme

ISOZYMES

Glycogen (%)

AMP (mm

4.5 18

0.044 0.095

0.035

6.9 18

0.1 0.18

0.11

hibited by higher concentrations of Na,SO,, although a slight stimulation was observed at a salt concentration of 0.1 M. In the absence of AMP, Na,SO, was slightly stimulative to the b forms of the isozymes, but was inhibitory to the a forms. Immunological

Studies

Antisera against purified muscle and liver phosphorylases yielded single precipitin lines against the respective specific antigens and crude tissue extracts on an agar plate (Fig. 4). No precipitin line was formed between anti-muscle phosphorylase antibody and the liver isozyme, and between anti-liver phosphorylase antibody and the a

IN THE

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muscle isozyme. On the other hand, a slight inhibition was observed when the muscle enzyme was treated with excess anti-liver enzyme antibody or when the liver enzyme was treated with excess anti-muscle enzyme antibody (Fig, 5). This suggests that two enzymes share at least one antigenic determinant in common. A mixture of the two antibodies completely inhibited the enzyme of spleen, kidney, and intestine, indicating that three isozymes detected in these organs would be identical with muscle and liver phosphorylases and their hybrid molecule. Antibodies were also slightly inhibitory to the phosphorylases of lamprey, crucian carp, and rainbow trout (Fig. 5 and Table IV). Amino

Acid Composition

Table V shows the results of amino acid analyses of skate muscle and liver phosphorylases, and also of lamprey phosphorylase. Although some differences were observed in the contents of lysine, histidine, arginine and alanine, the overall compositions of these poikilothermal phosphorylases were essentially similar. Assaf and Yunis (25) pointed out that the content of arginine was lower in poikilothermal muscle phosphorylases than in mammalian muscle

form

b

0,: /‘\

form

.

O-

-AMP

OF NagSO4

FIG. 3. Effects liver

phosphorylase;

of

/*::..= C---O

p----m.d I.11 LMI

3

.5

..a Y--/o .7

Na$O, on the a and b forms of skate phosphorylases. 0, muscle phosphorylase; solid lines, assayed with I mM AMP; broken lines, assayed without AMP.

0,

154

YONEZAWA

AND HORI

the muscle- and heart (or brain)-specific enzymes are active in the presence of AMP, (ii) the electrophoretic mobility differs between the skeletal muscle enzyme and heart-specific enzyme, (iii) the K, values for ligands of partially purified heart-specific phosphorylase b of crucian carp are lower than those of its muscle phosphorylase b (0.05 and 0.11% for glycogen, 0.02 and 0.12 FIG. 4. Ouchterlony agar diffusion plates. Plate A: mM for AMP, and 7 and 31 KIM for glucentral well, 20 ~1 of anti-skate muscle phosphorylcose l-phosphate, respectively). It is thus ase antiserum; wells 1-6, purified skate muscle phos- strongly suggested that functional differenphorylase b (1.2 mg/ml) serially diluted twofold (20 tiation of phosphorylase isozymes had al~1). Plate B: central well, 20 ~1 of antiskate liver phosready been accomplished during teleost phorylase antiserum; wells 1-3, purified skate liver evolution in the same direction as in higher phosphorylase b (0.5 mg/ml) serially diluted twofold and also that glycogen metab(20 ~1); wells 4-6, crude liver extract (4 and 6, 20 ~1; vertebrates, olism in elasmobranchs is not so fully dif5, 10 /LB. ferentiated as to require the presence of phosphorylases. This holds true in the case three specialized forms of phosphorylase. of lamprey and skate phosphorylases. DISCUSSION

Phosphorylase Isoxymes in Other Lower Vertebrates

Figure 6 shows the presence of three isozymes in the snake, toad, and crucian carp. Several lines of evidence indicate that these isozymes have characteristics quite similar to their mammalian counterparts. This evidence includes: (i) the liverspecific enzyme is characterized by the inactivity of its b form, while the b forms of

The present study has clearly demonstrated that skate possesses two different isozymes of phosphorylase, muscle and liver types. They differ in electrophoretic mobility, and kinetic and immunological properties, but were very similar in subunit molecular weight, amino acid composition, AMP dependence of the b form and sensitivity to SO:-. That their b forms are hybridizable may be taken as another evidence

A

“0

1

2 Antibody

3

L (~1 )

I

O 0

-’

‘-5

10 Antibody

15

20 ()Il)

FIG. 5. Inhibition by anti-skate muscle phosphorylase antibody (A) and anti-skate liver phosphorylase antibody (B) of phosphorylases from skate muscle (e), skate liver (0) and lamprey muscle (H). Each enzyme was incubated at 30°C with indicated amounts of antibody in a total volume of 0.05 ml. After 10 min, 0.45 ml of incubation medium was added and the remaining phosphorylase activity was assayed.

PHOSPHORYLASE

ISOZYMES

for their structural similarity. No evidence has been obtained that a third isozyme is present in skate tissues. Differences in kinetic properties between the muscle and liver isozymes may reflect different metabolic requirements between these tissues. Schliselfeld (5) has stated that phosphorylase I is the form adapted to tissues with a low concentration of AMP and moderate or higher concentrations of ATP and glucose 6-phosphate; phosphorylase L is advantageous for mammalian hepatic glycogenolysis which is hormonally controlled, since the absence of hormones yields catalytically inactive Lb and the presence of hormones yields catalytically active La; and phosphorylase III is suitable to such tissues as skeletal muscles where AMP and glycogen are present at high concentrations, since its b form has a low affinity to the ligands. With the same logic, it seems possible to say that elasmobranch muscle phosphorylase is a form specialized for muscular glycogen metabolism, since skate muscle isozyme has kinetic properties similar to mammalian isozyme III. In fact, similarities between elasmobranch and rabbit muscle phosphorylases have been reported by several workers (24, 25, 29, 30). TABLE RELATIVE

INHIBITION

IV

POTENCIES OF ANTIBODIES

Source of enzyme

Anti-skate muscle phosphorylase antibody

Anti-skate liver phosphorylase antibody

Skate Skeletal muscle Liver

1.00 0.01

0.03 1.00

Lamprey

0.01

0.07

Crucian carp Skeletal muscle Heart Liver”

0.03 0.11 0.01

0.04 0.07 0.01

Rainbow trout Skeletal muscle Heart

0.08 0.06

0.05 0.05

a Partially purified in the a form through DEAEcellulose column chromatography (9).

IN THE

155

SKATE TABLE

V

ANIINO ACID COMPOSITION OF PHOSPHORYLASES (males/94,000 g) Amino acid

Skate muscle

Skate liver

LYS His Aw Asp Thr Ser Glu Pro QY Ala Half-Cys Val Met Ile Leu Tyr Phe Trp

45 19 56 96 38 31 84 36 54 65 10” 96 il 25 56 67 38 38 13

61 27 42 100 39 33 80 35 49 55 12” 55 22 52 72 35 36 14

822

819

Total

Lamprey 56 26 51 99 35 32 80 35 48 64 10” 52 21 53 73 37 34 13 819

a Determined as cysteic acid. L Determined as free sulfhydryl groups in the SDSdenatured protein.

On the other hand, skate liver enzyme is distinctly different from the muscle isozyme, having lower affinities for AMP and glycogen, and also from mammalian isozyme L in that the b form of skate enzyme is active in the presence of AMP. Accordingly, the presence of such an isozymic form in the elasmobranch liver may be indicative of the absence of a hormonal control of glycogen metabolism and of high levels of glycogen and AMP in the elasmobranch liver. This inference is consistent with the finding of Patent (31) that in dogfish and ratfish the administration of adrenalin elicits no significant change in a tissue glycogen level. Idler et aE. (32) also reported that neither interrenalectomy nor hypophysectomy affected significantly the glycogen level in skate liver. From a comparison of relative inhibition potencies (RIP) of antilamprey phosphorylase antibody, we have previously predicted (13) that lamprey phosphorylase is more

156

YONEZAWA

M

H

B

L

AND HORI

between the skate isozymes is greater than that between lamprey phosphorylase and skate muscle or liver phosphorylase. Snake The present electrophoretic studies demonstrated that crucian carp, toad, and snake possess at least two AMP-dependent isozymes and one isozyme resembling mammalian phosphorylase L. It is highly probable that the muscle-, heart-, and liverspecific isazymes of these animals are comparable to mammalian isozymes III, I, and L, respectively. Consistent with this view is another finding that heart and skeletal muscle phosphorylase b of crucian carps show high and low affinities for AMP and glycogen, respectively, just as their mamFIG. 6. Diagrammatic representation of phosphorylmalian counterparts do. If this holds true, ase isozyme patterns of skeletal muscle (M), heart (H), one can even speculate that the duplicabrain (B), and liver (L) in snake, toad, and crucian tion event of phosphorylase gene had been carp. , isozymic forms, the b forms of which are actaken place two times before the separativated by AMP; =, isozymic form, the b form of tion of teleost fishes from the main vertewhich is inactive even in the presence of AMP. brate line leading to mammals, while one closely related to elasmobranch liver iso- duplication event of the gene had been zyme (RIP = 23%) than to elasmobranch taken place during elasmobranch evolution, muscle isozyme (RIP = 3%). In reciprocal assuming that a single molecular form in tests performed in the present study, RIP lamprey reflects the most primitive genetic of anti-skate liver phosphorylase antibody arrangement in ancestral vertebrates. The presence of an isozyme comparable with lamprey phosphorylase was 7% and that of anti-muscle phosphorylase antibody to mammalian isozyme L in crucian carp with lamprey phosphorylase was 1%. Al- liver might suggest that the teleost liver though the results of reciprocal tests did not has a highly specialized regulatory mechagree well, the data are not inconsistent anism of blood sugar level, such as that of with our prediction. Similarities in the af- mammalian liver. However, it has not been finities for ligands between lamprey and fully understood to what extent liver phosfor regulating skate liver phosphorylases also favor our phorylase is responsible view. blood sugar level in teleost fishes. Although Amino acid compositions of the muscle adrenalin and glucagon undoubtedly inphosphorylases from a wide variety of duce hyperglycemia in teleosts (35-3’7), animals have been reported to be very available data are conflicting with respect similar (11, 23-25, 30, 33). By contrast, to hepatic glycogen levels after the hormone rabbit liver phosphorylase appears to be injections; one reports the decrease (38), somewhat different from rabbit and rat and others, no change (39). In addition, muscle phosphorylases in amino acid com- there are some reports that question the contribution of hepatic glycogen to the position and in sequence of phosphorylated peptide (8, 9, 11). In the present study, regulation of blood sugar and of phosphocompositional relatedness has been clearly rylase to the degradation of glycogen in demonstrated among lamprey and skate teleosts; Chang and Idler (40) and Stimpson phosphorylases as judged from divergence (38) suggest that during fasting some factors of Harris et al. (34): 0.022 for the teleost fishes metabolize protein in preferlamprey versus the skate muscle; 0.021 for ence to glycogen and lipid, and Murat (41) the lamprey versus the skate liver; and suggests that y-amylase (EC 3.2.1.3) ap0.033 for the skate muscle versus skate pears to play a role in glycogen degradaliver. It is interesting that the difference tion induced in carp liver by insulin injec-

PHOSPHORYLASE

ISOZYMES

tions. Thus, the regulation mechanism of hepatic glycogen metabolism in teleosts is still a matter of debate, and the difficulty in solving this question may be related to an extensive adaptive radiation of teleosts. Studies on the isozymes concerning glycogen metabolism might be of some help in gaining an insight into this problem. ACKNOWLEDGMENTS We wish to thank Professor Koichi Yagi, Faculty of Science, Hokkaido University, and Dr. Tsugio Osugi, Faculty of Agriculture, Hokkaido University, for the permission to use some experimental facilities. We are also indebted to Dr. Keiji Kondo, Faculty of Agriculture, Hokkaido University, for his valuable guidance and advice in analyzing amino acid compositions of phosphorylases.

REFERENCES 1. YUNIS, A. A., FISCHER, E. H., AND KREBS, E. G. (1962) J. Biol. Chem. 237, 2809-2815. 2. DAVIS, C. H., SCHLISELFELD, L. H., WOLF, D. P., LEAVIW, C. A., AND KREBS, E. G. (1967) J. Biol. Chem. 242, 4824-4833. 3. HANABUSA, K., AND KOHNO, H. (1969) J. Biothem. (Tokyo) 66, 69-76. 4. SCHLISELFELD, L. H., DAVIS, C. H., AND KREBS, E. G. (1970) Biochemistry 9, 4959-4965. 5. SCHLISELFELD, L. H. (19’73) Ann. N.Y. Acad. Sci. 210, 181-190. 6. YONEZAWA, S., AND HORI, S. H. (1975) J. Histochem. Cytochem. 23, 745-751. 7. YONEZAWA, S., AND HORI, S. H. (1976) J. Biothem. (Tokyo) 79, 1109-1111. 8. APPLEMAN, M. M., KREBS, E. G., AND FISCHER, E. H. (1966) Biochemistry 5, 2101-2107. 9. WOLF, D. P., FISCHER, E. H., AND KREBS, E. G. (1970) Biochemistry 9, 1923-1929. 10. APPLEMAN, M. M., YUNIS, A. A., KREBS, E. G., AND FISCHER, E. H. (1963) J. Biol. Chem. 238, 1358-1361. 11. SEVILLA, C. L., AND FISCHER, E. H. (1969) Biochemistry 8, 2161-2171. 12. SEERY, V. L., FISCHER, E. H., AND TELLER, D. C. (1967) Biochemistry 6, 3315-3327. 13. YONEZAWA, S., AND HORI, S. H. (1977) Arch. Biochem. Biophys. 181, 447-453. 14. FISCHER, E. H., AND KREBS, E. G. (1958)5. Biol. Chem. 231, 65-71. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

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