Study on properties and regulation peculiarities of glycogenolysis enzymes in fish skeletal muscles depending on peculiarities of the species motor activity

The activity values, properties and peculiarities of activation of glycogen phosphorylase (GP, EC 2.4.1.1) and glycogen phosphorylase kinase (GPK, EC 2.7.138) were studied in the white skeletal muscle of fishes differing in motor behavior. No differences in the GP and GPK activity levels were revealed in porgy Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, trout Salmo trutta morphario, scorpionfish Scorpaena porcus, flounder Scophtalnus maeoticus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. The more rapid ion regulation prevails in fishes capable for the burst swimming type (scorpionfish, trout). The glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found in other species.     

Use of properties and regulation peculiarities of enzymes of glycogenolysis in fish skeletal muscle depending on peculiarities of motor activity of species

Levels of activity, properties, and peculiarities of activation of glycogen phosphorylase (GP; EC 2.4.1.1) and glycogen phosphorylase kinase (GPK; EC 2.7.1.38) were studied in the white skeletal muscle of fish differing in motor behavior. No differences in the GP and GPK activity levels were revealed in laskir Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, salmon Salmo trutta morphario, scorpena Scorpaena porcus, Scophtalnus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. In fish capable for the spurt type of swimming (scorpena, salmon) the more rapid ion regulation plays the predominant role. In other species, the glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Studies on phosphorylase b kinase from neutral tissues

Abstract— Phosphorylase b kinase (ATP: phosphorylase phosphotransferase; EC 2.7.1.38), the enzyme which converts phosphorylase b to phosphorylase a (α-1,4-glucan: orthophosphate glucosyltransferase; EC2.4.1.1) was examined in nerve tissue. Both phosphorylase and phosphorylase kinase were present in all nerve tissues tested, with central tissues about ten times as active as peripheral nerve. Exceptions were the superior cervical and stellate ganglia, tissues rich in synapses, which displayed activity similar to brain. Phosphorylase kinase in brain had properties similar to those of the enzyme in skeletal and cardiac muscle; it was activated in vitro by ATP and adenosine 3′,5′-monophosphate (cyclic AMP) and by Ca²⁺. Subconvulsive doses of insulin or of amphetamine administered to mice produced some activation of the enzyme. It is concluded that the mechanism for activation of phosphorylase in nerve tissue is similar to that in muscle.     

Seasonality of glycogen phosphorylase activity in crucian carp (<Emphasis Type="Italic">Carassius carassius</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Seasonal changes in the activity of glycogen phosphorylase (GP), a rate-limiting enzyme of glycogen degradation, were examined in an anoxia-tolerant fish species, the crucian carp (Carassius carassius L.). In muscle and brain, the activity of GP remained constant throughout the year when tested at 25°C. In contrast, the activities of liver and heart GP displayed striking increases in summer. When seasonal temperature changes are taken into account, the activity of GP during the anoxic mid-winter is only 4–6% of its summer time activity in the muscle, heart and liver, and 13% in brain. In winter-acclimatized fish, experimental anoxia (1–6 weeks) caused sustained depression of the GP activity in heart and gills. In liver and muscle, a transient depression of GP activity occurred during the first week of anoxia but later GP activity recovered back to the normoxic level. GP of the brain was completely resistant to anoxia. In all studied tissues, the constitutive activity of GP is more than sufficient to degrade glycogen deposits during winter anoxia without anoxia-induced activation of GP. The seemingly paradoxical summer-time increase in the activity of liver and heart GP could be related to active life-style of the summer-acclimatized fish (growth, reproduction), the increased demand of energy and molecular precursors of anabolic metabolism being satisfied by preferential degradation of glycogen. The high glycogen content of winter-acclimatized crucian carp is not associated with the elevated GP activity or anoxic activation of GP.     

Glycogen phosphorylase isoenzyme BB in diagnosis of myocardial ischaemic injury and infarction

This review deals with glycogen phosphorylase (GP) and its isoenzyme BB in the diagnosis of ischaemic myocardial injury. Early identification and confirmation of acute myocardial infarction is essential for correct patient care and disposition decision in the emergency department. In this respect, glycogen phosphorylase isoenzyme BB (GPBB) based on its metabolic function is an enzyme for early laboratory detection of ischaemia. In the aerobic heart muscle GPBB together with glycogen is tightly associated with the vesicles of the sarcoplasmic reticulum. Release of GPBB, the main isoform in the human myocardium, essentially depends on the degradation of glycogen, which is catalyzed by GP. Ischaemia is known to favour the conversion of bound GP in the b form into GP a, thereby accelerating glycogen breakdown, which is the ultimate prerequisite for getting GP into a soluble form being able to move freely in the cytosol. The efflux of GPBB into the extracellular fluid follows if ischaemia-induced structural alterations in the cell membrane become manifest. The clinical application of GPBB as a marker of ischaemic myocardial injury is a very promising tool for extending our knowledge of the severity of myocardial ischaemic events in the various coronary syndromes. The rational roots of this development were originated from Albert Wollenberger's research work on the biochemistry of cardiac ischaemia and the transient acceleration of glycogenolysis mainly brought about by GP activation.     

Effect of fasting on glycogen metabolism and activities of glycolytic and gluconeogenic enzymes in the mudskipper Boleophthalmus boddaerti

During starvation, muscle glycogen in Boleophthalmus boddaerti was utilized preferentially over liver glycogen. In the first 10 days of fasting, the ratio of the active‘a’form of glycogen phosphorylase to total phosphorylase present in the liver was small. During this period, the active‘I’form of glycogen synthetase increased in the same tissue. In the muscle, the phosphorylase‘a’activity declined during the first 7 days and increased thereafter while the total glycogen synthetase activity showed a drastic decline during the first 13 days of fasting. The glycogen level in the liver and muscle of mudskippers starved for 21 days increased after refeeding. After 6 and 12 h refeeding, liver glycogen level was 8·5 ± 2·3 and 6·9 ± 4·5 mg·g wet wt ¹, respectively, as compared to 5·8 ± l·6mg·g wet wt ¹ in unfed fish. Muscle glycogen level after 6 and 12 h refeeding was 0·96±0·76 and 0·82 ± 0·50 mg·g wet wt ¹, respectively, as opposed to 0·21 ± 0·12 mg·g wet wt ¹ in the 21-days fasted fish. At the same time, activities of glycogen phosphorylase in the muscle and liver increased while the active‘I’form of glycogen synthetase showed higher activity in the liver. Since glycogen was resynthesized upon refeeding, this eliminated the possibility that glycogen depletion during starvation was due to stress or physical exhaustion after handling by the investigator. Throughout the experimental starvation period, the body weight of the mudskipper decreased, with a maximum of 12% weight loss after 21 days. Liver lipid reserves were utilized at the onset of fasting but were thereafter resynthesized. Muscle proteins were also metabolized as the fish were visibly thinner. However, no apparent change in protein content expressed as per gram wet weight was detected as the tissue hydration state was maintained constant. The increased degradation of liver and muscle reserves was coupled to an increase in the activities of key gluconeogenic enzymes in the liver (G6Pase, FDPase, PEPCK, MDH and PC). The increase in glucose synthesis was possibly necessary to counteract hypoglycemia brought about by starvation in B. boddaerti.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

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.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

The swimming performance of brown trout and whitefish: the effects of exercise on Ca2 + handling and oxidative capacity of swimming muscles

The swimming performance of two fish species, the brown trout and whitefish, having initially different swimming strategies, was measured after nine different training programs in order to relate the effects of exercise on Ca²⁺ handling and oxidative capacity of swimming muscles. The time to 50% fatigue was measured during the training period, and compared with the density of dihydropyridine (DHP) and ryanodine (Ry) receptors and succinate dehydrogenase (SDH) and phosphorylase activity determined by histochemical analysis of the swimming muscles. Overall, both trained brown trout and whitefish had superior swimming performance as compared to control ones. Interestingly, the training programs had different effect on the two species studied since brown trout achieved the highest swimming performance, swimming against the water flow velocity of 2 BL s⁻¹ while among whitefish the best efficiency was seen after training with lower swimming velocities. Training also induced a significant increase in DHP and Ry receptor density in both species. Generally, in brown trout the most notable increase in the receptor densities was observed in red muscle sections from the fish swimming for 6 weeks against water currents of 1 BL s⁻¹ (DHPR 176.5 ± 7.7% and RyR 231.4 ± 11.8%) and white muscle sections against 2 BL s⁻¹ (DHPR 129.6 ± 12.4% and RyR 161.9 ± 15.5%). In whitefish the most prominent alterations were noted in samples from both muscle types after 6 weeks of training against water current of 1.5 BL s⁻¹ (DHPR 167.1 ± 16.9% and RyR 190.4 ± 19.4%). Finally, after all the training regimens the activity of SDH increased but the phosphorylase activity decreased significantly in both the species. To conclude, our findings demonstrate an improved swimming performance and enhanced Ca²⁺ regulation and oxidative capacity after training. Moreover, there seems to be a connection between the swimming performance and receptor levels, especially in white swimming muscles of different fish species, regardless of their initially deviant swimming behaviours. However, depending on the training regimen the divergent swimming behaviours do cause a different response, resulting in the most prominent adaptational changes in the receptor levels of red muscle samples with lower swimming velocities in brown trout and with higher ones in whitefish.     

Kinetics of Denaturation of Rabbit Skeletal Muscle Glycogen Phosphorylase b by Guanidine Hydrochloride

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity,an intermediate plateau,and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min^–1;1 M GuHCl, 25°C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.     

GLYCOGEN AND GLYCOGEN PHOSPHORYLASE IN THE CEREBRAL CORTEX OF MICE UNDER THE INFLUENCE OF METHIONINE SULPHOXIMINE

Abstract— Glucose and glycogen levels in the mouse cerebral cortex in vivo were studied after recovery from methionine sulphoximine seizures. The animals appeared normal 24 h after methionine sulphoximine administration but both glucose and glycogen still persisted at higher levels 72 h after injection (by 64 and 275 per cent, respectively). When seizures were prevented by methionine, the increase in glucose and glycogen at the longer time intervals was significantly smaller than in animals treated with methionine sulphoximine only; glucose reached normal values at 48 or 72 h; the accumulation of glycogen was reduced by about three to five times, but after 72 h the levels were still significantly higher than in control animals (67 or 32 per cent increase, depending on the administered dose of methionine). In contrast to the considerable accumulation of glycogen after administration of methionine sulphoximine in vivo, it had no effect on the level of glycogen in brain cortex slices in vitro. After 3 h incubation in the absence of methionine sulphoximine, glycogen was resynthesized to a level of about 4 μmol/g wet tissue and this value was not significantly affected by the presence of various concentrations of methionine sulphoximine in the incubation medium (10^-5 to 10^-2 M). The total (a+b forms) phosphorylase activity of mouse cerebral cortex in vivo after methionine sulphoximine administration was not affected. The fraction of active phosphorylase was reduced by about 50 per cent at the time of seizures. When seizures were prevented by methionine, the decrease in active phosphorylase was also completely prevented. In the preconvulsive period (1-2 h) and after recovery from the seizures (48 h after methionine sulphoximine administration) active phosphorylase was normal. The possible mechanisms involved in the increased accumulation of glycogen after methionine sulphoximine administration are discussed.     

Phosphorylase and gluco-6-phosphatase activity and glycogen level in tissues of vertebrates

The activity of phosphorylase (EC 2.4.1.1), glucose-6-phosphatase (EC 3.1.3.9) and the content of glycogen have been determined in tissues of fish, amphibians, reptiles, mammalians. No differences in the activity of phosphorylase and glucose-6-phosphatase in the liver, myocardium, and brain of animals of the phylogenetic groups under study are found. The activity of glucose-6-phosphatase in the anaerobic muscles of poikilothermal animals is found to be rather high. The share of phosphorylase a in the skeletal muscles and brain as well as the glycogen content in the brain of these animals is essentially higher than that of adult mammalians.     

Relationship of Glycogen Synthase and Glycogen Phosphorylase to Protein Phosphatase 2C and cAMP-Dependent Protein Kinase in Liver of Obese Rhesus Monkeys

ORTMEYER HK. Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys. The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/ threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determined. Basal enzyme activities and basal substrate concentrations were not significantly different between the three groups of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p<0. 05) and to PP2C activity (p=0. 005) (adjusted R²,53%). GP independent activity was correlated to GS independent activity (p<0. 07) and to PKA fractional activity (p=0. 005) (adjusted R²,64%). PP2C activity was correlated to GS fractional activity (p<0. 0005) and to PP1/2A activity G7<0. 0001) (adjusted R²,83%). PKA fractional activity was correlated to GP total activity (p<0. 0005) and to age (p=0. 001) (adjusted R282%). G6P content was correlated to glycogen content (p<0. 05) and to PP2C activity (p=0. 0005) (adjusted R²,73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.     

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10⁻⁴, 22 × 10⁻⁴, and 13 × 10⁻⁴m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.     

Factors responsible for glycogenolysis acceleration in early embryogensis of teleosts

Summary An acceleration of the rate of glycogenolysis in the early embryogenesis of the loach (Misgurnus fossilis L.) is accompanied by an increase of the content of hexose monophosphates, the rate of lactate formation and the rate of respiration. The unfertilized egg and the intact embryo of the loach have an identical activity of phosphorylase (EC 2.4.1.1.) and a constant ratio of the active/latent phosphorylase.Following the stage of 32 blastomeres, an increase of phosphorylase activity and the glycogen content occurs in the yolk-free embryo (blastoderm); this increase stops after the onset of gastrulation. In view of the facts that a) the blastoderm contains practically no latent phosphorylase, b) an elevation of phosphorylase activity is synchronized with an increase of the glycogen content, and c) this process is not related to an increase of the total phosphorylase activity and glycogen content in the intact egg, the authors suggest that glycogen-bound phosphorylase transfers from the yolk to the embryo at the stages of cleavage and blastula.In the loach oocyte, unfertilized egg and embryo the main activity of phosphorylase (more than 3/4) is associated with low molecular weight glycogen; this form of glycogen cannot be sedimented at 144000 g, and constitutes not more than 30 % of the total glycogen.Glycogen synthetase (EC 2.4.1.11) is, on the contrary, bound completely with granular glycogen. The oocyte maturation, ovulation and the onset of glycogenolysis after fertilization do not involve a redistribution of enzymes between glycogen fractions of different molecular weights.An increase of the glucose level in oocytes accelerates the conversion of active phosphorylase into its latent form. Physiological concentrations of glucose (up to 2 × 10^–2 M) do not inhibit phosphorylase activity.

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Zusammenfassung Ein unbefruchtetes Ei und ein intakter Schlammpeitzgerembryo (Misgurnus fossilis L.) weisen dieselbe Aktivität der Phosphorylase (EC 2.4.1.1) und ein konstantes Verhältnis zwischen der aktiven und der latenten Phosphorylase auf. Nach dem Stadium der 32 Blastomeren wurde im vom Eidotter isolierten Embryo (Blastoderm) eine Zunahme der Aktivität der Phosphorylase und die Zunahme des Glykogengehaltes festgestellt. Diese hört nach Beginn der Gastrulation auf. Die Verfasser begründen ihre These von der Übertragung der glykogengebundenen Phosphorylase aus dem Eidotter in den Embryo in den Phasen am Ende der Furchung und in der Blastula.In der Oozyte, im unbefruchteten Ei und im Embryo ist nur ein unbedeutender Teil der Phosphorylase mit dem granulären Glykogen verbunden. Die hauptsächliche Aktivität der Phosphorylase (etwa 80%) ist mit dem bei 144000 g nicht absetzbaren niedermolekularen Glykogen verbunden, das 30% des gesamten Glykogengehaltes nicht übersteigt. Die Glykogensynthetase (EC 2.4.1.11) ist umgekehrt mit dem granulären Glykogen verbunden. Das Reifen der Oozyte, die Ovulation und der Beginn der Furchung sind nicht in die Umverteilung der Fermente zwischen den Fraktionen des Glykogens verschiedenen Molekulargewichtes verwickelt.

     

Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B

This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5"-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5"-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.     

Purification of glycogen phosphorylase b from AMP-aminohydrolase activity

The presence of AMP aminohydrolase (EC 3.5.4.6) activity in glycogen phosphorylase b (EC 2.4.1.1) preparations, suggested by L. N. Johnson, N. B. Madsen, J. Mosley, and K. S. Wilson (1974, J. Mol. Biol.90, 703–717), has been confirmed in our laboratory. Since the hydrolase catalyzes the conversion of AMP into IMP the presence of traces of this impurity would dramatically affect, and could even invalidate, the results concerning some studies on the phosphorylase b-AMP interaction. The incubation of the phosphorylase b preparations with alumina Cγ in the cold for a brief period of time is proposed as a simple method of efficiently eliminating this impurity.