PURIFICATION AND MOLECULAR-PROPERTIES OF GLYCOGEN PHOSPHORYLASE-B FROM TROUT WHITE MUSCLE

Glycogen phosphorylase b (EC 2.4. 1. 1) was isolated from white skelet al muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the dire ction of glycogen breakdown at 21-degrees-C) by using glycogen - conca navalin A, DEAE-Sephadex, and 3,5'-cAMP affinity chromatography. Purif ied 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 mum, 31 mM and 142 muM, respecti vely, 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 inhibito rs; glucose inhibition decreased at lower pH values. IMP did not affec t the enzyme. The catalytic properties of trout phosphorylase b indica te that the enzyme would be virtually inactive at the physiological co ncentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, P(i), and AMP levels during b urst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolys is in working trout white muscle.