Inhibition Of Phosphorylase Phosphatase Research Articles

Regulation of glycogen phosphorylase and glycogen synthase by adrenalin in soleus muscle of phosphorylase-kinase-deficient mice.

Mixed skeletal muscles of ICR/IAn mice contain 0.2% of the phosphorylase kinase activity found in C3H/He-mg mice at pH 8.2 in the presence of Ca2+. This lack of activity is caused by the absence of the normal phosphorylase kinase protein (Cohen, P. T. W. et al. (1976) Eur. J. Biochem. 66, 347–356). The trace residual activity in ICR/IAn mice has quite different properties from the normal enzyme. It has a much higher activity ratio (pH 6.8/8.2), its activity is less dependent on Ca2+ and it is not activated by cyclicAMP-dependent protein kinase or by trypsin. Its elution behaviour on Sepharose 413 also differs from the normal enzyme. A consequence of the higher activity ratio (pH 6.8/8.2) and decreased effect of Ca2+, is that mixed skeletal muscles of ICR/IAn mice contain 7% of normal activity at pH 6.8 in the absence of Ca2+. The activity of phosphorylase kinase in C3H/He-mg control mice is sixfold lower in red oxidative (soleus) muscle and 15-fold lower in cardiac muscle than in mixed skeletal muscle. In contrast, the trace residual activity in ICR/IAn mice is present at the same level in soleus muscle, cardiac muscle and mixed skeletal muscle. Soleus muscles of ICR/IAn mice therefore contain 1.5% of normal activity at pH 8.2 in the presence of Ca2+ and 30% of normal activity at pH 6.8 in the absence of Ca2+. Cardiac muscle contains 6% of normal activity at pH 8.2 in the presence of Ca2+ and 60% of normal activity at pH 6.8 in the absence of Ca2+. When isolated soleus muscles were incubated with adrenalin, the level of phosphorylase a in ICR/IAn mice rose from 2.8% in the absence to 5.7% in the presence of the hormone. In contrast, the level of phosphorylase a in normal mice varied from 2.4% to 11.1% in the absence of adrenalin, and increased to 17.7–23.9% in the presence of the hormone. It is concluded that the trace phosphorylase kinase activity in ICR/IAn mice is capable of phosphorylating phosphorylase b in vivo and that this enzyme is responsible for the very low level of phosphorylase a in resting soleus muscle of ICR/IAn mice, and perhaps even in normal mice. Since the residual enzyme cannot be activated by cyclic-AMP-dependent protein kinase, the elevation of phosphorylase a by adrenalin may be caused by an inhibition of phosphorylase phosphatase. The possibility that this occurs through the activation of protein phosphatase inhibitor 1 by cyclic-AMP-dependent protein kinase is discussed. Incubation of soleus muscles with adrenalin decreased the activity ratio (+ glucose 6-phosphate) of glycogen synthase from 0.24 to 0.08 in ICR/IAn mice and 0.31 to 0.14 in C3H/He-mg or Swiss albino mice. These results demonstrate that the inactivation of glycogen synthase by adrenalin in resting soleus muscle does not result from the activation of phosphorylase kinase by cyclic-AMP-dependent protein kinase. It is most likely mediated by a direct phosphorylation catalysed by cyclic-AMP-dependent protein kinase, although the activation of protein phosphatase inhibitor 1 may also contribute to the effect.

Effects of chronic ethanol ingestion on male and female rat liver glycogen phosphorylase phosphatase.

The effects of chronic ethanol ingestion on the interconversion of the active to the inactive form of glycogen phosphorylase by phosphorylase phosphatase was studied. Male and female rats also were compared. Chronic ethanol feeding decreased phosphorylase a and total phosphorylase activity in male rats. In females, no change was observed in phosphorylase a, whereas total phosphorylase activity was increased 73%. This was found to correlate with the relative activities of phosphorylase phosphatase. The data show differences between the two sexes with regard to the AMP inhibition of phosphorylase phosphatase and the caffeine stimulation of the phosphatase. Ethanol markedly enhanced the AMP inhibition of the phosphatase in males but had no effect in females. Further studies in females showed that ethanol completely obliterated the well documented stimulation of the phosphatase by caffeine; however, it did not alter the caffeine effect in males. These data suggest possible alterations in the tertiary structure of phosphorylase a.

Inhibition of phosphorylase phosphatase by heat-stable inhibitor proteins of dog liver

Inhibition of phosphorylase phosphatase by heat-stable inhibitor proteins of dog liver

Autonomic neural control of liver glycogen metabolism

Neural and humoral induction of hepatic glycogenolysis occurs via different pathways. Sympathetic nerve stimulation activates alpha adrenergic receptors to produce what is probably a cyclic AMP independent, Ca ++ dependent activation of glycogen phosphorylase. Evidence suggests that phosphorylase ‘a’ is activated by inhibition of phosphorylase phosphatase and perhaps by activation of phosphorylase kinase. Blood borne catecholamines act via beta adrenergic receptors to produce a cyclic AMP dependent activation of phosphorylase kinase. Additionally, hepatic parasympathetic nerves can produce a dramatic shut-down of hepatic glucose output by activation of glycogen synthetase and inhibition of glycogen phosphorylase. The observed minor effect of insulin or intravenous glucose on hepatic glucose uptake compared to the effect of oral doses of glucose suggests that an additional factor other than insulin can produce hepatic glucose uptake: this factor could be, and probably is, the hepatic parasympathetic nerves. The present hypotheses result from a combination and extrapolation of whole animal physiological and in vitro biochemical data.

Inhibition of phosphorylase phosphatase by polyamines

Phosphorylase phosphatase from rabbit liver or from bovine heart, purified as the M r 35,000 form, is inhibited by the naturally occurring polyamines, spermine and spermidine. The inhibition is apparently competitive with a K i of 0.037 mM for spermine and 0.65 mM for spermidine. Further investigation showed that the inhibition is substrate-directed, i.e. due to an interaction of the polyamines with the rabbit muscle phosphorylase a used as the substrate.

Role of Phosphorylase Kinase and Cyclic AMP-dependent Protein Kinase in the Regulation of Phosphorylase Phosphatase

The enzymes of glycogen metabolism are regulated by interconversion between phosphorylated and dephosphorylated forms. In contrast with our well-defined knowledge of the phosphorylation processes of different enzymes and their role in glycogen breakdown and synthesis, the phosphatases catalysing 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 . l) into inactive phosphorylase b by cleavage of the phosphate groups from the serine residues. For the control of phosphorylase phosphatase (EC 3.1.3.17) some mechanisms are possible, namely ligands that affect the substrate phosphorylase a or phosphatase; proteins that affect phosphorylase a (or phosphatase); or enzymic modification of phosphatase. It is known that different proteins can influence the phosphorylase phosphatase reaction. The activation of phosphorylase with synchronous inhibition of phosphorylase phosphatase was observed in the protein-glycogen complex isolated from rabbit skeletal muscle (Haschke et af., 1970). No clear explanation has been offered for the transient inhibition of phosphorylase phosphatase during the ‘flash activation’. Haschke et af. (1972) attributed the inhibition to a protein-protein interaction caused by an unknown protein component of the complex. This assumption was supported by frontal gel filtration of muscle extract and purified enzymes showing a strong association between phosphorylase, phosphorylase kinase and phosphatase (Gergely et al., 1974, 1975). A heat-stable protein isolated from different mammalian tissues also inhibits the phosphatase reaction (Brandt et af., 1974, 1975~). This heat-stable inhibitor protein is phosphorylated by cyclic AMP-dependent protein kinase (Cohen rt al., 1977). Toth et al. (1977) demonstrated the reversible phosphorylation and dephosphorylation of this heat-stable inhibitor protein in vivo. Huang & Glinsmann (1975, 1976a,h) demonstrated the existence of two heat-stable inhibitor proteins for phosphatase. In the present study the effect of phosphorylase kinase and cyclic AMP-dependent protein kinase on the regulation of phosphorylase phosphatase has been investigated. Rabbit skeletal-muscle phosphorylase a was prepared and assayed as previously described (Bot et al., 1977). The specific activity of phosphorylase a was 55 units/mg in the presence of 0.016~-gh1cose 1-phosphate and in the absence of AMP. Non-activated phosphorylase kinase was prepared from rabbit skeletal muscle, and its activity was measured, as described by Cohen (1973). Thiophosphate-activated phosphorylase kinase was prepared by the method of Gergely et al. (1976). Cyclic AMP-dependent protein kinase was prepared from rabbit skeletal muscle by the method of Beavo e ta / . (1974). The separation of regulatory and catalytic subunits of protein kinase was carried out on a Blue Dextran/Sepharose column (Witt & Roskoski, 1975). The isolated protein kinase holoenzyme, the separated regulatory and catalytic subunits, phosphorylase a and phosphorylase kinase were homogeneous by sodium dodecyl sulphate (0.1 %)/ polyacrylamide-gel (7.5 %) electrophoresis. The gel-electrophoresis experiments were carried out as described by Weber & Osborn (1969). Phosphorylase phosphatase was prepared by method of Brandt et a/. (197%). Inhibition of the phosphorylase phosphatase reaction by phosphorylase kinase and cyclic AMP-dependent protein kinase was assayed in the following manner. Phosphorylase a (1 .Omg/ml) was incubated in 0.04~-Tris/2m~-EDTA/O.5m~-dithiothreito~ buffer (pH7.4) with phosphorylase phosphatase in the presence of various concentrations of phosphorylase kinase or cyclic AMP-dependent protein kinase or its regulatory/ catalytic subunit. The reaction mixtures were incubated at 30T , portions (50~1) were removed at various times, and the reaction was stopped by the addition of 0.1 M-NaF/

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.

The influence of ATP on glycogen synthetase D phosphatase activity in liver

1. 1. A number of compounds from pathways associated with glucose and glycogen metabolism have been evaluated as regulators of the liver synthetase D phosphatase reaction. Although several were found to be inhibitory to the reaction, only ATP was an effective inhibitor at physiological concentrations. 2. 2. The inhibition by ATP was not a manifestation of either a stimulation of synthetase kinase or phosphoryllase kinase or the inhibition of phosphorylase phosphatase. All of these effects could result in an apparent inhibition of synthetase phosphatase. Whether the effect of ATP is on synthetase phosphatase or synthetase D, its substrate, is as yet unknown.

Effect of the administration of the antischistosomal drug niridazole on muscle glycogen levels of monkeys.

Abstract Niridazole was administered to 57 rhesus monkeys at a dosage schedule approximating the minimally curative dose for schistosomiasis mansoni of this species. This resulted in a marked depletion of the muscle glycogen stores, which was attributed in part to a reduced food intake due to anorexia produced by the drug and in part to an inhibition of phosphorylase phosphatase, activity bringing about an accumulation of active glycogen phosphorylase. Restoration of muscle glycogen levels to control values was observed about 8 days after discontinuation of drug administration.