A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis.
We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992). We have examined the effects of the reaction components on the catalytic activities of both "primary" and "coupling" enzymes. While MTGuo exhibits no effect on the glycogen phosphorylase-catalyzed reaction, glucose 1-phosphate and AMP are partially inhibitory to nucleoside phosphorylase. However, the latter effects pose no problem as long as the coupling enzyme is maintained at a relatively higher concentration in the assay system. The coupled enzyme assay system, standardized for the measurement of glycogen phosphorlase activity, has enabled us to demonstrate explicitly that the rate of the enzyme-catalyzed reaction exhibits sigmoidal dependence on both AMP and glucose 1-phosphate concentrations. We argue that these sigmoidal profiles have been observed due to the sensitivity and precision of the present assay system.
Sergienko
E A
EA
Biochemistry Department, North Dakota State University, Fargo 58105.
Srivastava
D K
DK
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003460:Crystallization; D005958:Glucosephosphates / Q000494:pharmacology; D006003:Glycogen / Q000096:biosynthesis*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010763:Phosphorylase b / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012044:Regression Analysis; D013053:Spectrophotometry / Q000379:methods; D013997:Time Factors
D005958:Glucosephosphates; D000249:Adenosine Monophosphate; D006003:Glycogen; C031590:glucose-1-phosphate; D010763:Phosphorylase b