METABOLIC CONTROL AND STRUCTURE OF GLYCOLYTIC ENZYMES .8. REVERSAL OF DISSOCIATION OF RABBIT MUSCLE PYRUVATE KINASE INTO UNFOLDED SUBUNITS

The dissociation of tetrameric rabbit muscle pyruvate kinase (mol wt 237,000) into unfolded subunits (mol wt 57,000) in 6 m guanidine hydrochloride has been shown to be reversible. A systematic study of the factors affecting the reversal of the dissociation led to conditions where up to 70 % of the initial catalytic activity was regained. The reversal procedure (Deal, W. C., Jr. (1969), Biochemistry (in press)) consisted of two phases: (1) a 100-fold dilution of guanidine hydrochloride dissociated enzyme (0°) into a reversal solvent at 0° and (2) incubation of the resulting solution at a higher temperature, usually 16°. Conditions for optimum reversal of dissociation were (1) pH 8; (2) protein concentration, 0.04 mg/ml; (3) ionic strength, 0.3; (4) reducing agent, 0.06 β smercaptoethanol; and (5) temperature, 0° dilution, followed by 6 hr at 16°. The half-time for activity recovery was approimately 45 min at both 0.02 and 0.09 mg per ml enzyme concentration. Two metabolites, insulin and phosphate ion, were found to greatly influence the reversal of dissociation. Insulin decreased the activity recovery upon reversal, in contrast to what would be expected for an inducer of the enzyme. Phosphate ion yielded activity recovery at 36°; negligible activity was recovered at that temperature in its absence. The reversal of dissociation was not affected significantly by the addition of a number of metabolites including adenosine triphosphate, adenosine diphosphate, 5′-adenosine monophosphate, 3′,5′- adenosine monophosphate, lactate, fructose diphosphate, and nicotinamide-adenine dinucleotide. The reassociated enzyme had the same Km, heat stability, and sedimentation coefficient as the native enzyme. © 1969, American Chemical Society. All rights reserved.