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.     

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.     

Effect of polycarboxylates on phosphorylase b.

Activation of phosphorylase b by AMP is stimulated by certain aliphatic and cyclic polycarboxylates. This stimulation was depended on the number and the position of the carboxyl groups, the stereochemistry and the size of the molecule, and was more pronounced at low AMP concentrations. Kinetic studies indicated that in the presence of polycarboxylates the affinity of the enzyme for AMP was enhanced, the cooperative binding of the nucleotide was removed, and the enzyme was no longer inhibited by glucose-6-phosphate. Although polycarboxylates have no effect on the sedimentation pattern of phosphorylase b in the absence of AMP, the partial association of the enzyme caused by AMP is greatly enhanced in the presence of the acids.     

Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase.

It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.     

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.     

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.     

Histochemical studies on phosphorylase activity in the tissues of the albino rat under normal and experimental conditions

Summary Various substrate mixtures have been tested for the histochemical demonstration of phosphorylase in tissue blocks. These studies indicate that phosphorylase activity, cytological detail and localization of the reaction product are optimally demonstrated when tissue blocks are incubated for one hour or longer in a medium containing high concentrations of G-1-P, ATP and PVP.Abbreviations AMP adenosine-5-monophosphate - ATP adenosine-5-triphosphate - EDTA ethylenediamine-tetraacetic acid - G-1-P glucose-1-phosphate - PPa active phosphorylase - PPb inactive phosphorylase - PVP polyvinyl pyrrolidone     

Spin label studies on phosphorylase B.

Spin label studies upon phosphorylase B have revealed that the temperature-dependent equilibria between different conformational states are strongly influenced by the presence of substrate or allosteric modifier. In the absence of substrate or modifier, the equilibrium involves only two states, while at least three are involved in their presence. Removal of pyridoxal-5′-phosphate produces a conformational change reflected by increased mobility of the label. The apo-enzyme undergoes a structural change in the presence of the allosteric modifier AMP.     

Regulation of platelet phosphorylase.

A sensitive fluorimetric enzyme assay was developed for study of activation of glycogen phosphorylase (EC 2.4.1.1) in intact platelets and in platelet extracts. Activity was calculated as AMP independent (activity in the absence of AMP), total (activity in the presence of 1 mM AMP), and AMP dependent (difference between AMP independent and total). The following observations were made with intact rat platelets. (1) Stimulation of platelets with thrombin caused a 7-fold increase in total activity, with increases in both AMP-dependent and AMP-independent activities. Maximum activation was obtained within 10 s after addition of thrombin. (2) The divalent cation ionophore A23187 caused a similar, though less pronounced, activation of phosphorylase. (3) Acceleration of glycogenolysis by inhibition of respiration with cyanide caused similar changes in phosphorylase activity but with the maximum effect observed only after 45 s. (4) Dibutyryl cyclic AMP had two effects; it partially activated phosphorylase and blocked further activation by thrombin, but not A23187. Similar effects were observed with human platelets, but low resting levels of phosphorylase activity could not be maintained so that changes were not as large as with rat platelets. Experiments with extracts of rat platelets gave the following results. (1) Phosphorylase activity in many extracts of non-stimulated platelets could be increased by incubation with Mg2+-ATP and Ca2+; ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) partially inhibited. (2) In some extracts there was essentially no activation by incubation with Mg2+-ATP and Ca2+, but addition of cyclic AMP GAVE PARTIAL ACTIVATIon while addition of rabbit muscle phosphorylase kinase gave full activation. (3) Incubation of extracts of thrombin-stimulated platelets caused conversion of AMP-dependent to AMP-indeptndent activity. It is concluded that platelet phosphorylase exists in an inactive and two active forms. Conversion of the inactive to the active forms and of the AMP-dependent to the AMP-independent form is catalyzed by a kinase(s) that requires Ca2+ for full activity and is activated through a cyclic AMP-mediated process. The major change following physiological stimulation is an increase in both active forms, with little change in their ratio.     

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.     

Effect of temperature on the kinetics of the phosphorylase reaction and phosphorylase activation induced by phosphorylase kinase in the skate Dasyatis pastinaca

The dependence of the phosphorylation reaction rate on the glucose-1-phosphate concentration is investigated in Dasyatis pastinaca in a wide temperature range (5-45 degrees C). In the temperature range of 20-40 degrees C nH is equal to 1.3-1.7. The disturbance of allosteric interactions of active sites with the loss of kinetic substrate cooperativity is observed at 45 degrees C. v(S)-Dependence with the intermediate plateau is obtained at 5 degrees C and high concentration of glycogen phosphorylase B (EC 2.4.1.1), that is explained by the formation of inactive tetramer. Studies in activation of glycogen phosphorylase B of Dasyatis pastinaca under the effect of glycogen phosphorylase (EC 2.7.1.38) kinase have revealed temperature-dependent changes in the pattern of kinetic curve. An assumption is advanced that the presence of the association-dissociation equilibrium in oligomeric forms of glycogen phosphorylase B with different enzymic activity and the effect of the temperature-dependent conformation state of this enzyme on the kinase reaction rate plays an essential role in regulation of glycogenolysis in the muscular tissue of ectothermal animals.