Citrate synthase in the rainbow trout heart: regulation by pH, temperature, and metabolite levels

FERGUSON, R. A., and STOREY, K. B. 1991. Citrate synthase in the rainbow trout heart: regulation by pH, temperature, and metabolite levels. Can. J. Zool. 69: 3020-3027. Crude homogenates of rainbow trout (Oncorhynchus mykiss) hearts have high citrate synthase activity (30 units per gram wet weight at 20°C), reflecting the aerobic nature of this organ. Citrate synthase was purified 70-fold by polyethylene glycol fractionation followed by ion-exchange separation on phosphocellulose and DEAE columns. At 20°C the partially purified enzyme displayed hyperbolic Michaelis-Menton-type substrate kinetics and an activity maximum at pH 8.5. The K, values for oxaloacetic acid and acetylcoenzyme A were 1.65 and 3.91 kM, respectively. Competitive inhibition with respect to oxaloacetic acid was seen with citrate (Ki = 8.80 mM) and a-ketoglutarate (Ki = 13.95 mM). Noncompetitive inhibition by ATP (Ki = 1.04 mM) with respect to oxaloacetic acid was observed. ATP (Ki = 0.382 mM) and NADH (Ki = 0.45 1 mM) were competitive inhibitors for acetylcoenzyme A. Citrate synthase activity is both pHand temperate-dependent. Indeed, the temperature dependence (0-25°C) is modified by pH. This is reflected by a decrease in the activation energy from 15.7 to 10.6 kcaYmol(65.63 to 44.30 Hlmol) as the pH of the medium is increased from 7.05 to 7.80. These findings are discussed with respect to trout heart citrate synthase function in vivo.