Abstract
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 Km values for oxaloacetic acid and acetylcoenzyme A were 1.65 and 3.91 μM, respectively. Competitive inhibition with respect to oxaloacetic acid was seen with citrate (Ki = 8.80 mM) and α-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 = 0.451 mM) were competitive inhibitors for acetylcoenzyme A. Citrate synthase activity is both pH- and 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 kcal/mol (65.63 to 44.30 kJ/mol) 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.
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