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.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001667:Binding, Competitive; D002110:Caffeine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010762:Phosphorylase a / Q000037:antagonists & inhibitors / Q000378:metabolism; D010763:Phosphorylase b / Q000037:antagonists & inhibitors / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits
D005958:Glucosephosphates; D007291:Inosine Monophosphate; D002110:Caffeine; D000249:Adenosine Monophosphate; D019298:Glucose-6-Phosphate; C031590:glucose-1-phosphate; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases; D005947:Glucose