Walter H. Glinsmann

A second heat-stable protein inhibitor of phosphorylase phosphatase from rabbit muscle

Glycogen phosphorylase phosphatase (EC 3.1.3.17) may be a hormonally-regulated enzyme since its activity in tissue extracts is controlled by a cyclic AMP-mediated reaction [ 1,2] . However, attempts to characterize the regulation of this enzyme have been difficult since the enzyme can be isolated in multiple molecular forms [3,4], appears to have broad substrate specificity [5,6], and loses the ability to be regulated by cyclic AMP and the cyclic AMP-dependent protein kinase when it is purified [7,8]. One possibility for regulation is that the enzyme contains a catalytic subunit which can dephosphorylate many proteins and that regulatory subunit(s) restrict catalytic attack and are also the site of cyclic AMPstimulated phosphorylations. With both liver and muscle, relatively heat-stable phosphatase inhibitor proteins can be isolated from larger molecular weight proteins containing phosphatase activity [9, IO]. Inactivation of the rabbit muscle enzyme by cyclic AMP-dependent protein kinase is associated with phosphorylation of one of these proteins [lo]. The phosphorylated inhibitor is separable from the phosphatase by sucrose gradient centrifugation or gel permeation chromatography. However, following such separation, heat-stable inhibitor activity remains associated with the phosphatase. We now provide evidence that this residual activity is due to a second protein inhibitor of the phosphatase which does not require phosphorylation for activity. 2. Methods

Activation of glycogen synthetase and inactivation of phosphorylase kinase by the same phosphoprotein phosphatase.

Abstract Purified rabbit muscle glycogen synthetase D phosphatase inactivates phosphorylase kinase. The inactivation is reversed by cyclic AMP-dependent protein kinase. It is postulated that the synthetase D phosphatase is a general phosphoprotein phosphatase which dephosphorylates proteins that are phosphorylated in vivo by the cyclic AMP-dependent kinase.

Glycogen Regulation in Isolated Perfused Near Term Monkey Liver

Extract: Glycogen metabolism was studied in the isolated perfused liver of the monkey conceptus at 90% of gestation using an in situ recirculating perfusion system. Net uptake of glucose and galactose and the activities of the enzymes, glycogen synthetase and phosphorylase, were studied in response to varied perfusate composition. Synthetase activity was expressed as %1, the percentage of total synthetase activity in the active form. Perfusate glucose concentrations as high as 700 mg/100 ml did not lead to net glucose uptake or to an increase in the baseline %I synthetase (4 ± 1, mean ± SEM). In the presence of 300 mg/100 ml glucose, insulin at 10-7 M increased %I to 8 ± 2, and galactose > 75 mg/100 ml increased %I to 8 ± 1. The combination of galactose, glucose, and insulin increased %I to 40 ± 5. With this latter combination, synthetase activity was proportional to perfusale glucose concentration above 100 mg/100 ml. Phosphorylase activity was diminished by either galactose or insulin, and phosphorylase activity was lowest in the group receiving galactose, glucose, and insulin. Galactose was taken up by all livers, but net glucose uptake was not observed under any condition; net hexose uptake was observed in perfusions with galactose. Glycogen levels did not vary significantly with varied perfusate composition during the 30-min perfusion periods.Speculation: We speculate that galactose may be uniquely important for neonatal liver glycogen synthesis, and that its action is mediated through reciprocal changes in the activities of the enzymes, glycogen synthetase and phosphorylase. If liver glycogen is important for acute neonatal glucose homeostasis, then galactose may also be essential for maintaining circulating glucose concentration by ensuring glycogen synthesis during feeding.

Regulation of glycogen synthetase activity by two kinases

Summary Rabbit muscle glycogen synthetase was found to be phosphorylated both by cyclic AMP-dependent protein kinase and by a cyclic AMP-independent protein kinase that also phosphorylates phosvitin. Phosphorylation by either kinase alone resulted in only partial conversion of synthetase to a glucose-6-P-dependent form. More complete conversion could be achieved when phosphorylation by phosvitin kinase preceeded phosphorylation by cyclic AMP-dependent protein kinase, but not when the order of phosphorylation was reversed. A novel type of regulatory mechanism involving the interaction of these two kinases with three phosphorylation sites is postulated.

Multiple forms of protein phosphatase inhibitors in mammalian tissues

Abstract The heat-stable inhibitors of phosphorylase phosphatase from rat skeletal muscle, rat liver, beef heart, and beef adrenal cortex were studied. Two major types of inhibitors can be separated from all these tissues by chromatography on DEAE-cellulose. The two types of inhibitors from rat muscle are the same as those reported for rabbit muscle. The type-1 inhibitor, but not the type-2, is regulated by phosphorylation and dephosphorylation reactions. There is also a phosphorylatable inhibitor in beef heart and adrenal cortex, but such an inhibitor could not be demonstrated in liver.

Regulation of rat liver glycogen synthesis and activities of glycogen cycle enzymes by glucose and galactose

Direct regulation of rat liver glycogen metabolism by glucose and galactose was studied using an isolated liver perfusion system. Activation of glycogen synthase and net glycogen synthesis increased linearly when perfusate glucose concentration was increased from 125 to 500 mg/100 ml. Galactose, rapidly taken up by isolated rat liver regardless of circulating glucose concentration, increased these responses to glucose. In the presence of galactose (greater than or equal to 5 mg/100 ml), activation of synthase and glycogen synthesis were 1.5-fold higher at any given glucose concentration. The addition of insulin did not appreciably after synthase activation by glucose and galactose. Phosphorylase activity, low at circulating glucose levels above 125 mg/100 ml, was further decreased as glucose was increased or when galactose was added to the perfusate. Release of glucose into the perfusate in response to aglycemia was increased in the presence of galactose.

Effect of limited proteolysis on activity and phosphorylation of rabbit muscle glycogen synthetase.

Abstract Purified rabbit skeletal muscle glycogen synthetase, in both the glucose-6-phosphate (P)-dependent (phosphorylated) and the glucose-6-P-independent (dephosphorylated) forms, was subjected to limited proteolysis by trypsin. Both forms could be degraded from their original subunit molecular weight of 85,000 to 76,000 and subsequently to 68,000, as determined with acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Degradation of the glucose-6-P-dependent form of the enzyme resulted in essentially no change in the activity when measured either in the presence or in the absence of glucose-6-P. Degradation of the glucose-6-P-independent form was associated with a progressive increase in glucose-6-P dependency. Phosphorylation of the glucose-6-P-independent form with the adenosine 3′,5′-monophosphate-dependent protein kinase and subsequent digestion of the 32 P-labeled enzyme showed that the phosphate group was retained on these subunits. The protein kinase phosphorylated both the original subunit with molecular weight 85,000 and the partially digested subunit with molecular weight 76,000. Upon further digestion of the enzyme into a form having a subunit molecular weight of 68,000, the enzyme was unable to accept a phosphate group from ATP. By contrast with the phosphorylation reaction, the dephosphorylation reaction catalyzed by partially purified glycogen synthetase phosphatase is not stringent in terms of structural integrity of the synthetase. The phosphatase dephosphorylated the glucose-6-P-dependent form of glycogen synthetase equally well at various degrees of degradation.

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine.

SummaryThe effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (Ki value of 0.3mm) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylasea.

Partial purification of the glucocorticoid receptor from rat liver: A rapid, two-step procedure using DNA-cellulose

Abstract Glucocorticoid receptor from rat liver was purified 1800-fold by a rapid two-step procedure using DNA-cellulose. The procedure is based on increasing the affinity of the glucocorticoid-receptor complex for DNA by heating the complex. During a first chromatography step, unheated glucocorticoid-receptor complex is separated from cytosol proteins that bind to DNA-cellulose with high affinity. During a second chromatographic step, heat-treated glucocorticoid-receptor complex is separated from proteins with low affinity for DNA. The partially purified complex is functionally competent in that it is taken up by isolated rat liver nuclei.

Effects of polyamines on nucleosidediphosphate kinase activity

Abstract Effects of naturally occurring polyamines were tested on the activity of bovine liver nucleosidediphosphate kinase with ATP as phosphoryl donor and eight nucleosidediphosphates as phosphoryl acceptors. The enzyme was either stimulated, inhibited, or unaffected depending upon the nucleosidediphosphate substrate and the polyamine, indicating that a general cation effect is not a sole mechanism of polyamine action. This selectivity and specificity of the effects with respect to both the polyamines and the nucleosidediphosphates leads us to speculate that an action of polyamines on nucleosidediphosphate kinase may play a significant role in the regulation of specific nucleosidetriphosphate synthesis in vivo .