Purification and molecular properties of glycogen phosphorylase b from trout white muscle.
Glycogen phosphorylase b (EC 2.4.1.1) was isolated from white skeletal muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the direction of glycogen breakdown at 21 degrees C) by using glycogen--concanavalin A, DEAE-Sephadex, and 3',5'-cAMP affinity chromatography. Purified phosphorylase b was a dimer with a native molecular weight of 193,000 and a subunit molecular weight of 87,000. Michaelis constants for glycogen, phosphate, and AMP were 128 microM, 31 mM and 142 microM, respectively, at pH 7.2; maximum activity of the enzyme was obtained at pH 7.5 and 25 degrees C. Glucose and ATP behaved as phosphorylase b inhibitors; glucose inhibition decreased at lower pH values. IMP did not affect the enzyme. The catalytic properties of trout phosphorylase b indicate that the enzyme would be virtually inactive at the physiological concentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, Pi, and AMP levels during burst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolysis in working trout white muscle.
Mehrani
H
H
Department of Chemistry, Carleton University, Ottawa, ON, Canada.
Storey
K B
KB
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002384:Catalysis; D002845:Chromatography; D005947:Glucose / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D017686:Oncorhynchus mykiss; D010710:Phosphates / Q000378:metabolism; D010763:Phosphorylase b / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D010710:Phosphates; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D006003:Glycogen; D010763:Phosphorylase b; D005947:Glucose