Electrophoretic studies on the phosphorylase isozymes.

The electrophoretic method of Davis, Schliselfeld, Wolf, Leavitt and Krebs (1967) for phosphorylase isozymes has been modified. By this method, five isozymes were separated in various organs of rat and pig and were disignated as phosphorylase L, LI, I, II and III. The L and III enzymes were the only forms found in liver and skeletal muscle, respectively, while the I enzyme was dominant in brain, uterus, lung and small intestine, which also contained some fractions of the II and III enzymes. The I enzyme was also dominant in adrenal, ovary and kidney, but these organs contained the L+II or L+LI as minor components. The L and LI were richly found in spleen and leukocytes of adult rats and pigs and in liver of newborn rats. Such organ-specific heterogeneity of phosphorylase was confirmed by the immunological tests with the antibodies prepared against phosphorylases I, III and L. The II and LI enzymes were found to be the hybrid molecules between the I and III enzymes, and between the I and L enzymes which have been previously reported as unhybridizable, respectively. In view of the above findings, it was concluded that the rat and pig possessed at least five molecular forms of phosphorylase.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase

1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.     

Nicotinamide riboside phosphorylase from beef liver: purification and characterization

Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.     

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.     

Separation and properties of three forms of cathepsin H-like cysteine proteinase from rat spleen

Three forms of cathepsin H-like cysteine proteinase were purified from rat spleen by a method involving acid treatment and chromatography on pepstatin-Sepharose, Sephadex G-75, DEAE-Sephacel, CM-Toyopearl, and concanavalin A-Sepharose. The final preparations of these forms all migrated as single protein bands on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS). The molecular weights of the three forms were estimated to be 28,000 (form I), 26,000 (form II), and 22,000 (form III). The optimal pH was 6.5 for forms I and III and was 7.0 for form II with L-leucine 2-naphthylamide (Leu-NA) or with alpha-N-benzoyl-DL-arginine 2-naphthylamide (BANA). All of the forms consisted of two major species having isoelectric points of 7.1 and 6.5 on isoelectric focusing gels. They were all stable when incubated at pH values between 5.0 and 9.0 for 1 h at 22 degrees C. They were strongly inhibited by iodoacetic acid and E-64, but not by metal ions or pepstatin. Form III was not affected by leupeptin, chymostatin, antipain or elastatinal, which gave essentially complete inhibition of cathepsin B purified from rat spleen. Forms I and II were slightly inhibited by these compounds at the same concentrations. The properties of these forms were compared with those of the known enzymes cathepsin H and BANA-hydrolase.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Antiserum against the catalytic subunit of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases.

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.     

Microheterogeneity of rat glycogen phosphorylase liver-type isozyme

We devised a method of polyacrylamide gel electrophoresis at pH 7.3, modified by omitting base catalyst, N,N,N',N'-tetramethylethylenediamine, in the preparation of separating gels. Using this method, both liver and liver-like types of rat glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) were resolved into multiple forms, about 6-10, although either of them was purified to a single protein with the same molecular size on sodium dodecyl sulfate gel electrophoresis. The microheterogeneity of these two types was also confirmed by isoelectric focusing in polyacrylamide gels (pH 5-8). The major isoelectric points of the liver type phosphorylase were between 5.72 and 5.86, but those of the liver-like type were between 5.86 and 5.92, and so the former had slightly but significantly lower isoelectric points than the latter. However, the both types were not distinguished immunologically. The brain and muscle types of rat phosphorylase did not show such a distinct heterogeneity by the same electrophoresis methods.     

Studies on phosphorylase isozymes in lower vertebrates. Purification and properties of lamprey phosphorylase.

Skeletal muscle phosphorylase b has been purified from lamprey, Entosphenus japonicus, to a state of homogeneity as judged by the criterion of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme was completely dependent on AMP for activity and converted into the a form by rabbit muscle phosphorylase kinase in the presence of ATP and Mg²⁺. The subunit molecular weight determined by SDS-gel electrophoresis was 94,000 ± 1,600 (SE). The enzyme activity was stimulated by Na₂SO₄, but was not affected by mercaptoethanol. The K_m values of the a form for glucose 1-phosphate and glycogen were 3.5 mm and 0.13%, respectively, and those of the b form for glucose 1-phosphate, glycogen, and AMP were 15 mm, 0.4%, and 0.1 mm, respectively. These values were smaller than those reported with lobster phosphorylase and greater than those reported with mammalian skeletal muscle phosphorylases. Electrophoretic and immunological studies have indicated that lamprey phosphorylase b exists as a single molecular form in skeletal muscle, heart, brain, and kidney. Rabbit antibody against lamprey phosphorylase cross-reacted with phosphorylases from skate and shark livers more intensely than with those from skeletal muscles.     

Some properties of starch phosphorylase from cotyledons of germinating seeds of Voandzeia subterranea.

Two isoenzymes (Forms I and II) of starch phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) were found in cotyledons of germinating seeds of Voandzeia subterranea L. Thouars. Phosphorylase I, which was the major component, had a pH optimum of 5.5--5.6, whereas phosphorylase II had a pH optimum of 6.1--6.3. Phosphorylase I had a molecular weight of 204 000 +/- 4000 and a subunit molecular weight of about 95 000. Phosphorylase I was stimulated by Mg2+, Mn2+, AMP, cyclic AMP, pyruvate and EDTA, but inhibited by Fe2+, Cu2+, Zn2+ and ATP. Stimulation of phosphorulase I by AMP was accompanied by changes in the affinity of the enzyme for glucose-1-phosphate in the presence of increasing AMP concentrations, and of AMP in the presence of increasing glucose-1-phosphate concentrations. Double-reciprocal plots of initial velocity data were non-linear (convex up) at low glucose-1-phosphate concentrations but became linear in the presence of AMP or ATP. Double-reciprocal plots were linear at high glucose-1-phosphate concentrations in the absence or presence of modifiers.     

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

Two distinct phosphorylase isozymes, skeletal muscle phosphorylase b and liver phosphorylase b, 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 SO₄^2?. 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.     

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Activation and inactivation of glycogen phosphorylase isoenzymes purified from diabetic rat heart

1. Hearts of diabetic rats gradually accumulate glycogen, although the activities of glycogen synthase and glycogen phosphorylase are altered in favor of a depletion of glycogen. 2. Phosphorylase in diabetic hearts has been reported to be even more activated in response to adrenaline than controls. 3. The situation is further complicated by the fact that in rat heart two isoenzymes of phosphorylase are present. Therefore we have studied the properties of phosphorylases purified from diabetic rat heart in more detail. 4. This investigation revealed that compared to controls: (A) the amount of enzyme protein which could be isolated from diabetic animals is drastically lower; (B) the affinities towards glycogen and inorganic phosphate are decreased; (C) the activation by phosphorylase kinase is delayed; and (D) the inactivation by protein phosphatase-1 is accelerated. 5. We conclude that all of the reported changes in diabetes might contribute to a phosphorylase system less able to catalyze glycogen breakdown effectively.     

Demonstration of 5'-methylthioadenosine phosphorylase activity in various rat tissues. Some properties of the enzyme from rat lung.

An enzyme (5'-methylthioadenosine phosphorylase) that catalyzes the phosphorolytic cleavage of 5'-methylthioadenosine to 5-methylthioadenosine to 5-methylthioribose-1-phosphate and adenine was found in various rat tissues. Liver and lung had the highest enzyme activities and heart the lowest, most of the activity (greater than 90%) was recovered in soluble tissue fractions. The enzyme from rat lung was purified about 30-fold by pH treatment (NH4)2SO4 fractionation, and gel filtration. The enzyme did not require an added metal-ion for activity, and was not inhibited by EDTA. Many compounds were tested for their inhibitory effects; of these, ribose 1-phosphate, 2-deoxyribose 1-phosphate, fructose 1-phosphate, adenine and guanine were shown to inhibit. Kinetic patterns on reciprocal plots were linear as a function of the concentration of either 5'-methylthioadenosine or phosphate. More detailed kinetic studies suggested that the rat lung 5'-methylioadenosine phosphorylase catalyzes an equilibrium-ordered reaction, and that 5'-methylthioadenosine is the first substrate to bind and 5-methylthioribose-1-phosphate is the first product to be released.     

Characterization of two different genes (cDNA) for cytochrome c oxidase subunit VIa from heart and liver of the rat.

By antibody screening of a rat liver and a rat heart cDNA library in lambda gt11 two clones coding for the liver- and heart-specific subunit VIa of rat cytochrome c oxidase were isolated. In the heart cDNA sequence a TAA stop codon was found in frame 18 bp 5' upstream of the first methionine codon, thus excluding a leader sequence for this protein. The two cDNAs contain the full-length coding region of two subunits. The amino acid sequences of the two subunits show only 50% homology, whereas 74% homology was found between rat heart and bovine heart subunit VIa. By Northern blot analysis it is shown that the gene for subunit VIa from heart is only expressed in heart and skeletal muscle, whereas that from liver is also expressed in kidney, brain, heart and weakly in muscle.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

Purification and characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada: amino acid sequence of the phosphorylation site

1. Skeletal muscle glycogen phosphorylase b has been purified from Liza ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be 97,000. By gel filtration a relative Mr of 190,000 was found. 3. Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a [32P]-labelled peptide which is observed to contain Ser, its sequence being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of mullet phosphorylase, 32P was incorporated in different protein bands resolved by isoelectric focusing. The degree of radioactivity associated with each one changed with the incubation time.