Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum

Abstract

Aerobic and anoxic variants of pyruvate kinase (termed PK-aer and PK-anx) from the hepatopancreas of the gastropod mollusc, Busycotypus canaliculatum, were purified to apparent homogeneity with final specific activities of 14 and 2.3 units/mg protein, respectively. Both enzymes were homotetramers of the same molecular weight. The enzymes also showed equivalent affinities for ADP (0.22 m m) and very similar affinities for Mg 2+, Mn 2+, K +, and NH 4 +. PK-aer and PK-anx differed strongly, however, in maximal enzyme velocity ( V max 9-fold higher for PK-aer), in affinity for P-enolpyruvate (PEP 0.5 = 0.38 m m for PK-aer and 1.1 m m for PK-anx), and in the effects of activators and inhibitors on the enzymes. PK-aer was much more strongly stimulated by fructose-1,6-P 2 and aspartate as activators (a 19- and 32-fold activation of enzyme velocity at sub-saturating PEP levels versus only 4.1- and 2.6-fold activation for PK-anx, respectively). K a for fructose-1,6-P 2 was 3-fold lower (0.16 μ m) for PK-aer than for PK-anx (0.48 μ m), but K a for aspartate was the same for both enzymes (1.5 m m). Activators decreased the PEP 0.5 (to 0.05 m m for PK-aer and 0.07 m m for PK-anx), relieved inhibitions by alanine, Mg ATP, ADP, and P i, and, when added together, showed a strong synergistic activation of PK-aer (but not PK-anx). The kinetic differences between PK-aer and PK-anx are similar to those of the dephosphorylated versus phosphorylated forms of PK from other sources, including those of red muscle PK of B. canaliculatum, and indicate that the change in enzyme form brought about during anaerobiosis may be due to enzyme phosphorylation. The powerful activation of hepatopancreas PK by aspartate is a novel regulatory control of the enzyme. Aspartate is one of the substrates of anaerobic energy production in marine molluscs and its effects on the enzyme may be important in a tissue where inactivation of PK can occur for one of two reasons: anaerobiosis or gluconeogenesis.