Enzyme on glycogen

Whelan and Roberts have devised an alternative method involving the successive action of salivary a-amylase and R-enzyme on glycogen. -By determining the number of reducing groups produced by action of R-enzyme on the a-dextrins, the proportion of (1 6) linkages can be calcu-... 

This ester was first isolated as a crude product by Cori, Colowick, and Cori 218) as a result of the action of frog muscle enzymes on glycogen and inorganic phosphate. A pure product was obtained when rabbit muscle extracts were dialyzed and then incubated with glycogen, phosphate buffer, and adenylic acid. The latter served as an activator of the enzyme (phos-phorylase). 

Fig. 7.21. Activation of glycogen-bound protein phosphatase I by insulin. Insulin has a stimulating effect on glycogen synthesis by initiating the dephosphorylation and activation of glycogen synthase and the dephosphorylation and inhibition of glycogen phosphorylase. Both enzymes (substrate S in the figure) are dephosphorylated by protein phosphatase PPIG. Insulin mediates the activation of a protein kinase (insulin-sensitive protein kinase) within an insulin-stimulated signal pathway, which phosphorylates and thus activates protein phosphatase PPIG at the PI site.

The extent and specificity of the reactions of protein kinases and protein phosphatases are extremely dependent on the degree to which substrate and enzyme are localized at the same place in the cell. Many substrates of protein kinases occur either as membrane associated or particle associated forms (see 7.6.1, enzymes of glycogen metabolism). For protein kinases or protein phosphatases to perform their physiological function in a signal transduction process, they must be transported to the location of then-substrate in many cases (review Hubbard and Cohen, 1992 Mochly-Rosen, 1995). This is vahd both for the Ser/Tbr-specific protein kinases as well as for many Tyr-speci-fic protein kinases. In the course of activation of signal transduction pathways, com-partmentahzation of protein kinases, redistributed to new subcellular locations, is often observed. 

The dephosphorylated form of the carboxylase does not require citrate for activity, but the phosphorylated form of the enzyme can be activated by citrate in vitro. This reaction is reminiscent of the effect of glucose-6-phosphate on glycogen synthase. The active, dephosphorylated form of glycogen synthase has only a small requirement for glucose-6-phosphate, whereas high concentrations of this activator are required to activate the phosphorylated form of glycogen synthase. 

Earlier studies on the properties of phosphorylases isolated from various sources have indicated that their subunits are similar in size with about 100,000 daltons.15-17 The reaction proceeds in a rapid equilibrium random Bi-Bi mechanism as has been shown by kinetic studies with rabbit skeletal muscle phosphorylases a18-20 and b,21,22 rabbit liver enzyme,23 potato tuber enzyme,24 and the enzyme from E. coli.25) In contrast, the substrate specificities for various glucans differ considerably depending on the enzyme sources. The rabbit muscle enzyme has high affinity for branched glucans such as glycogen and amylopectin but low affinity for amylose and maltodextrin.26,27 The potato tuber enzyme can act on amylose, amylopectin, and maltodextrin but only poorly on glycogen,28,29 while the E. coli enzyme shows high affinity for maltodextrin.10 ... 

Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the following defects caused by mutation (a) loss of the cAMP-binding site on the regulatory subunit of protein kinase A (PKA) (b) loss of the protein phosphatase inhibitor (inhibitor 1 in Fig. 15-40) (c) overexpression of phosphorylase b kinase in liver (d) defective glucagon receptors in liver. 

Vats, V., Yadav, S.P. and Grover, J.K. (2003) Effect of Trigonella foenum-graecum on glycogen content of tissues and the key enzymes of carbohydrate metabolism. Journal of Ethnopharmacology 85(2/3), 23 7-242. 

Murphy SD, Porter S. 1966. Effects of toxic chemicals on some adaptive liver enzymes, liver glycogen, and blood glucose in fasted rats. Biochem Pharmacol 15 1665-1676. 

Glycogen phosphorylase b stock solution—Dissolve 25 mg ( 750 units) of glycogen phosphorylase b (Sigma catalog P-6635) in approximately 750 pi of concentrated enzyme storage buffer (resuspend the lyophilized protein gently, do not use a vortex mixer). Divide into 20 pi aliquots and store at —20°C. Dilute this 1000-unit/ml stock solution to the required concentration (see protocol) just before use. Store the dilute enzyme on ice until it is ready to be used. 

The plant and bacterial enzymes capable of hydrolyzing pullulan do not have identical specificities. In particular, the plant enzymes have little or no action on glycogen and phytoglycogen under conditions in which they readily hydrolyze amylopectin and its /3-dextrin. To stress this difference (the bacterial enzymes are capable of degrading both glycogen and phytoglycogen), Manners (1997) recommended different nomenclature for bacterial enzymes, to be called pullulanase, and the plant enzymes, to be called limit dextrinases. 

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