Purification and Properties of Adductor Muscle Phosphofructokinase from the Oyster, Crassostrea virginica

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Abstract
Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 μmol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 ± 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 or pH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8. the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate. NAD(P)H, Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K+, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP (02–1.0 mM) and to a lesser extent by high concentrations of P, (20 mM) and aqdenosine 3′:5′-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki(ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8. whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulation of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.