György Bot

Heparin inhibits the activity of protein phosphatase-1.

Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase‐1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin—Sepharose CL‐6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor‐1. The phosphatase bound to heparin—Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor‐1.

Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin- Sepharose

Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. The latent enzyme had an absolute polycation (histone H1, polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted in the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontaneously active phosphatases were differentiated according to their abilities to dephosphorylate the alpha and the beta subunits of phosphorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-1 phosphatases, respectively.

The control of phosphorylase phosphatase by cAMP-dependent protein kinase.

The enzymes of glycogen metabolism are regulated by interconversion between phosphorylated and dephosphorylated forms. Contrary to our well-defined knowledge of the phosphorylation processes of different enzymes and their role in glycogen breakdown and synthesis, the phosphatases catalyzing the reverse processes are only now being understood. The regulation of dephosphorylation reactions is still unclear. Phosphorylase phosphatase converts ‘active’ phosphorylase a (EC 2.4.1.1) into ‘inactive’ phosphorylase b by cleavage of the phosphate groups from the serine residues. For the control of phosphorylase phosphatase the following mechanisms are possible: (i) By ligands which affect the substrate phosphorylase a (or phosphatase). (ii) By proteins which affect phosphorylase a (or phosphatase). (iii) By enzymatic modification of phosphatase. It was reasoned that ligands (e.g. glucose, glucose 6_phosphate, glycogen, AMP and caffeine) influence phosphorylase phosphatase reaction by changing the conformation of the substrate phosphorylase a or by shifting the tetramer _ dimer equilibrium of phosphorylase a [l-8]. It is known that different proteins can mediate the phosphorylase phosphatase reaction. We have shown that phosphorylase kinase inhibits the dephosphorylation of phosphorylase a and the inhibition is competitive in nature [9]. The phosphorylation (or thiophos; phorylation) of phosphorylase kinase increases its inhibitor constant [lo]. Another protein isolated from different mammalian

Partial phosphorylation of muscle phosphorylase: II. Formation of a hybrid phosphorylase in vivo

Abstract The conversion of rabbit skeletal muscle phosphorylase b (1,4-α- d -glucan: orthophosphate α-glucosyltransferase, EC 2.4.1.1) to phosphorylase α by phosphorylase b kinase has been investigated. In agreement with previous reports it was established that during the interconversion a partially phosphorylated hybrid phosphorylase is formed. Hybrid phosphorylase was detected both by the incorporation of 32P from γ-labelled ATP and by a new assay of phosphorylase activity. This assay is based upon the observation that the activity of hybrid phosphorylase is equally increased by AMP in the presence or the absence of caffeine, whereas AMP-induced activity of phosphorylase b is inhibited by caffeine. The extent of phosphorylation is controlled by the relative amount of phosphorylase b and of phosphorylase b kinase. Increasing the concentration of phosphorylase b or decreasing the concentration of phosporylase b kinase lead to the formation of hybrid phosphorylase.

Hormonal regulation of phosphorylase phosphatase activity in rat liver

The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat‐stable phosphatase inhibitors in the hormone‐treated samples. Liver protein kinases (regulatory subunit of cAMP‐dependent protein kinase and/or Ca2+ ‐dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).

Effect of fructose 1-phosphate on the activation of liver glycogen synthase

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.

Regulation of glycogen phosphorylase in Drosophila melanogaster by reversible phosphorylation-dephosphorylation

Abstract Homogeneous glycogen phosphorylase b purified from Drosophila melanogaster was activated by phosphorylase kinase isolated from rabbit skeletal muscle. The activation generated phosphorylase a containing 1.1 ± 0.1 phosphoryl group per subunit. Phosphorylase a prepared in this way had a s 20w = 8.5S and a subunit molecular mass of 95,000. It could be inactivated (dephosphorylated) by the catalytic subunit of rabbit muscle protein phosphatase-1. The activation-inactivation of phosphorylase by endogenous phosphorylase kinase and phosphatase was also demonstrated in crude homogenates of D. melanogaster . The main protein phosphorylated in the fruit fly homogenate comigrated with purified Drosophila phosphorylse a in sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Phosphate incorporation into this protein was correlated with phosphorylase activation. Phosphorylase was partially purified from flies fed on [ 32 P]phosphate by 5′-AMP Sepharose affinity chromatography. SDS gel electrophoresis followed by autoradiography revealed that it was phosphorylated in vivo . 20 ± 5% of the total phosphorylase was found to be in the active a form in the anaesthetized insects. Our findings indicate that Drosophila phosphorylase undergoes reversible phosphorylation-dephosphorylation.

Effects of acidic and basic macromolecules on the activity of protein phosphatase-1

The dephosphorylation of phosphorylase a by the catalytic subunit of protein phosphatase-1 obtained from rabbit skeletal muscle is inhibited by heparin in a noncompetitive manner with respect to phosphorylase a (Ki = 8 micrograms/ml). The inhibitory effect of heparin is also observed in the presence of effectors (e.g., glucose and AMP) modifying the dephosphorylation of phosphorylase a. Heat-stable protein inhibitors of protein phosphatase-1 can develop their inhibitory effect of the activity of protein phosphatase-1 even in the presence of heparin. The inhibitory effect of heparin and the heat-stable inhibitor-2 of phosphatase is additive. Polybrene, a heparin antagonist, prevented phosphatase-1 from the inhibition caused by heparin or the inhibitors. Proteins with basic character, histone fractions (H1, H3) and protamine sulfate, can counteract with the inhibitory effect of heparin, but they cannot intercept the actions of inhibitor-1 or -2.

Purification and characterization of glycogen phosphorylase from Drosophila melanogaster

Glycogen phosphorylase b was purified from Drosophila melanogaster with a yield of 15% by low speed centrifugation, DEAE-Sepharose CL-6B chromatography and affinity chromatography on 5′-AMP-Sepharose 4B. The preparation proved to be homogeneous by SDS-polyacrylamide gel electrophoresis and showed a subunit molecular weight of 95,000. Gel filtration of the native enzyme on Sephacryl S-300 revealed a molecular weight of 176,000 suggesting that phosphorylase b is a dimer composed of two identical subunits. The fruit fly phosphorylase b has a maximal specific activity of 36 U/mg when assayed in the direction of glycogen synthesis. It requires AMP for activity (A0.5 = 0.4 mM, Hill coefficient: 1.8). The KM for glycogen is 0.4% and the S0.5 for glucose-1-phosphate is 20 mM (Hill coefficient: 1.3). The enzyme is inhibited by glucose, UDPG, caffeine, glucose-6-phosphate, ATP and IMP.

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.