A primer independent activity of rabbit muscle phosphorylaseb

Summary Rabbit muscle phosphorylaseb was found to be capable of forming protein bound±-1,4 glucosyl chains upon incubation of the enzyme with appropriate concentrations of glucose-1-phosphate with no primer addition (unprimed synthesis). This activity would only be present in a small fraction of the total muscle phosphorylaseb activity, as judged from the high concentrations of enzyme which are required to demonstrate the occurrence of unprimed synthesis. Polyacrylamide gel electrophoresis shows the presence of a phosphorylase isoenzyme capable of accepting glucosyl moieties, giving rise to a glucosylated protein enzymatically active in the chain lengthening of its own glucan.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

Escherichia coli K-12 glycogen synthase: ability to use UDPglucose and ADP glucose as glucosyl donors in the absence of added primer.

A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Inhibition of alpha-glucan phosphorylase by alpha-D-glucopyranosyl fluoride.

Alpha-D-Glucopyranosyl fluoride was found to inhibit strongly the action of alpha-glucan phosphorylase b[EC 2.4.1.1] from rabbit muscle, and that of the enzyme from potato tubers rather weakly. The inhibition is highly specific, being competitive with respect to glucose 1-phosphate and noncompetitive with respect to polysaccharide, during polysaccharide synthesis. In the reverse process, it is competitive with respect to Pi. These results have been explained by assuming that the inhibitor binds to the glucose 1-phosphate site of the enzyme, occupying both subsites which normally bind the glucosyl and phosphate moities of the substrate, but does not directly interact with the polysaccharide site. Based on this assumption, the dissociation constants of the enzyme-inhibitor and enzyme-polysaccharide-inhibitor complexes have been evaluated (0.43 and 0.20 mM for the muscle enzyme, respectively; 24 and 23 mM for the potato enzyme, respectively). Glucosyl fluoride also acts as a noncompetitive inhibitor with respect to AMP. A high concentration of AMP causes an inhibitory effect on the action of the muscle enzyme, the effect being menifested in the presence of glucosyl fluoride.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

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.     

De novo Synthesis of Polyglucans by a Phosphorylase Isoenzyme in Algae

The α2 phosphorylase isoenzyme of Oscillatoria princeps, Rhodymenia pertusa and Chlorella pyrcnoidosa has been found to be capable of the synthesis of amylose-like polyglucans without the necessity for addition of primers. This enzyme differs in this respect from the a1 isoenzyme of blue-green and green algae. The a2 enzyme appears to be a glyco-protein and synthesis of linear polyglucans appears to occur by the apposition of glucosyl residues from glucose-1-phos-phate directly to the glycosyl moiety of its molecule.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.     

Phosphorylase kinase from chicken gizzard. Partial purification and characterization

Phosphorylase kinase was partially purified (530-970-fold) from chicken gizzard smooth muscle by a procedure involving ammonium sulfate fractionation, chromatography on 8-(6-aminohexyl)adenosine-5'-phosphate--Sepharose 4B and glycerol density gradient ultracentrifugation. The final and most efficient purification step takes advantage of the relatively high molecular mass of gizzard phosphorylase kinase, which was found to be similar to that of rabbit skeletal muscle enzyme. The gizzard kinase, further purified to near homogeneity by calmodulin-Sepharose 4 B affinity chromatography, showed one main protein band of 61 kDa, upon dodecyl sulfate acrylamide gel electrophoresis. Four minor protein bands of higher molecular mass were also present but no protein stain was seen at the position of the gamma subunit. The gizzard phosphorylase kinase showed a high pH 6.8/8.2 activity ratio of 0.53, it was stimulated by Ca2+, inhibited up to 80% by EGTA and it was activated about 1.9-fold by calmodulin. The km value for ATP was 0.45 mM, while the K0.5 for rabbit muscle phosphorylase b was extremely low, more than 200-fold lower than the Km of nonactivated skeletal muscle phosphorylase kinase for its protein substrate. High concentrations of phosphorylase b were found to be inhibitory. At 10 mg/ml phosphorylase b, the maximum activity of the kinase was inhibited fivefold. No evidence has been obtained indicating autophosphorylation or the existence of active and inactive forms of gizzard phosphorylase kinase. Limited proteolysis of the smooth muscle kinase with trypsin was accompanied by a twofold activation at pH 6.8.     

Cellobiose phosphorylase from Cellulomonas uda: gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a ‘glycosynthase-type’ reaction

We have cloned and sequenced the gene encoding cellobiose phosphorylase from Cellulomonas uda and report high yield production in Escherichia coli of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide. Use of heterologous gene expression increases the space-time yield of active phosphorylase by three orders of magnitude, compared to production of the enzyme with the natural organism. The full-length phosphorylase is a 91.3 kDa protein that consists of 821 amino acids and whose primary structure shares significant residue identity with different members of glycosyltransferase family 36. Purified enzyme was obtained in 39% overall yield by using copper-chelate and hydroxyapatite chromatographies. A comparative steady-state kinetic analysis for enzymatic reactions in the directions of phosphorolysis and synthesis of cellobiose at 30 °C and pH 6.6 demonstrates that the catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase. The ability of the phosphorylase to utilize - -glucose 1-fluoride (G1F) as alternate glucosyl donor in place of - -glucose 1-phosphate (G1P) is exploited for the synthesis of β-1,4-glucosides under thermodynamic control in close to 100% yield.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Inactivation and reactivation of liver phosphorylase b kinase.

When crude rat liver preparations were incubated at 30degrees C, a gradual loss of phosphorylase kinase (ATP:phosphorylase b phosphotransferase, EC 2.7.1.38) activity was observed. This inactivation was Mg2+ dependent and was partially inhibited by sodium fluoride. Addition of Mg2+ ATP to the liver preparations, at any time throughout the incubation, caused a reactivation of the phosphorylase kinase and this was accelerated by micromolar concentrations of cyclic AMP. The reactivation process could be completely abolished by the addition of a heat stable protein kinase inhibitor, implicating cyclic AMP dependent protein kinase in the activation reaction. Both the low and the high activity forms of the enzyme required micromolar quantities of Ca2+ for full activity (KA = 0.6 micronM). The two forms exhibit quite different pH dependencies and at the physiological pH of liver (pH 7.4) their activities differed by a factor of 5-10. Conversion of the lower activity form into the higher seems to affect only the V - Km for muscle phosphorylase b (EC 2.4.1.1) was about 1 mg/ml for both enzyme forms.     

Modification of rabbit muscle phosphorylase b by a water-soluble carbodiimide.

Glycogen phosphorylase b from rabbit muscle was rapidly inactivated by incubation with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) at pH 5.1. The inactivation was pH-dependent and was not restored by treatment with hydroxylamine. The addition of glycine ethyl ester or N-(2,4-dinitrophenyl)-ethylenediamine (DNP-EDA) markedly increased the rate of inactivation. Of the various amino analogs of glucose tested, only glucosyl amine accelerated the inactivation, although they are all bound to the glucose 1-phosphate site of the enzyme. In the absence of amines, incorporation of about 3 mol of [metho-14C]CMC per protein monomer was observed on complete inactivation. In the presence of DNP-EDA, however, only 2 mol of [metho-14C]CMC and 1 mol of DNP-EDA were incorporated before the activity was completely lost. The treatment of phosphorylase b with CMC did not change the Km values of the enzyme for glucose 1-phosphate and AMP, in spite of the 56% inactivation. It is suggested that, in the phosphorylase-catalyzed reaction, an essential carboxyl group of the enzyme plays a role in the protonation of the glucosidic oxygen of glucose 1-phosphate.     

Phosphorylase b covalently bound to glycogen: properties of the complex

Rabbit skeletal muscle glycogen phosphorylase b was covalently bound to oyster glycogen by means of cyanogen bromide. Removal of the unbound enzyme was achieved, using DEAE-Sephadex A-50 chromatography. Glycogen-bound phosphorylase b showed a higher affinity toward glucose 1-phosphate but a lower homotropic cooperativity, with respect to AMP activation, than the native enzyme. However, at low AMP concentrations conjugated phosphorylase b was as efficient as the free enzyme. It is of interest that glycogen-bound phosphorylase b exhibited catalytic activity upon its polysaccharide carrier. Kinetics of heat and cold inactivation indicated that the bound enzyme was considerably more resistant toward heat inactivation but less stable upon exposure to cold. It was shown also that both conjugated and native enzymes had identical pH optima, similar activity/temperature dependencies and the same resistance against trypsin inactivation.     

Inverse relationship of skeletal muscle glycogen from wild-type and genetically modified mice to their phosphorylase a activity.

Leg muscle was biopsied and frozen for storage at -70 degrees C. from 5 wild-type mice, two knocked out acid alpha-glucosidase (GAA) gene mice, and seven glycogen synthase plus glucose muscle transporter transgenic mice. All of the wild-type mice had very little muscle glycogen (3.58 +/- 1.67 micromols glucosyl subunits per g muscle), and 52% or more of its glycogen phosphorylase activity without AMP (69% +/- 17% glycogen phosphorylase a). In contrast the GAA knockout and transgenic mice had glycogen ranging from 63 to 297 micromols glucosyl subunits per g muscle, and very little or no glycogen phosphorylase activity without 1.00 mM AMP (4.8% and less glycogen phosphorylase a). This suggests that there is an inverse relationship between mouse muscle phosphorylase a and the muscle's glycogen content.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Crystallization of pig skeletal phosphorylase b. Purification, physical and catalytic characterization

A new method for purification and crystallization of pig skeletal muscle phosphorylase b is presented. The ease of crystallization in the presence of 1 mM AMP and 1 mM spermine has permitted the study of some physical, chemical and enzymatic properties of the enzyme. The crystalline pig phosphorylase b gave a single band on SDS polyacrylamide gels of the same mobility as rabbit muscle phosphorylase subunit. Ultracentrifugation experiments showed that pig phosphorylase b exists in a dimeric form (S20,w = 8.4 S). No association occurred at 20 degrees C under conditions where rabbit phosphorylase b can be tetramerized; pig phosphorylase b was only 30% associated from dimer to tetramer at 13 degrees C. Pig phosphorylase b is highly stable to freezing and its specific activity did not change appreciably upon prolonged storage in the cold. Pig and rabbit phosphorylases b have comparable Vmax and Km values towards the substrate and the activator. However, there is an essential difference between the two enzymes in that pig phosphorylase b is not significantly inhibited by glucose 6-phosphate, which is a powerful inhibitor of the rabbit enzyme. Two different crystal forms of pig phosphorylase b were obtained which are small for X-ray diffraction studies. Diffusion of spermine into tetragonal crystals of rabbit phosphorylase b resulted in a difference Fourier synthesis at 3 A resolution that showed no strong indication of specific binding.     

An engineered liver glycogen phosphorylase with AMP allosteric activation

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Changes in the activity of enzymes,participating in glycogen metabolism of alloxan diabetic rats

Summary Alloxan diabetes induced in white rats by intraperitoneal injection of Aloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished.AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities.The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.