Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum.

Abstract

Aerobic and anoxic variants of radular retractor muscle pyruvate kinase (PK-aerobic and Pk-anoxic) from the gastropod mollusc, Busycotypus canaliculatum, were purified to homogeneity and respective specific activities of 368 and 186 mumol of product min-1 mg protein-1. Both PK variants were apparent homotetramers with native molecular masses of about 235 kDa, but differed in several other physical characteristics including pI (5.81 +/- 0.06 for PK-aerobic, 5.42 +/- 0.03 for PK-anoxic) and chromatographic behavior on several columns used during their respective purifications. The two enzymes differed greatly in several kinetic properties. Affinity for phosphoenolpyruvate was more than tenfold greater for PK-aerobic (K0.5 = 0.067 +/- 0.002 mM; h = 0.99 +/- 0.10), whereas the cooperative effect for phosphoenolpyruvate binding was greatly enhanced for PK-anoxic (K0.5 = 0.85 +/- 0.02 mM, h = 2.57 +/- 0.01). Although the affinities for the second substrate, ADP, were identical for both enzyme forms (apparent Km = 0.25 mM) pK-anoxic showed greater substrate inhibition by high concentrations of ADP. Likewise, affinities for K+ and Mg2+ were similar but PK-anoxic showed a greater degree of cooperativity with Mg2+ (h = 2.50 +/- 0.02) than did PK-aerobic (h = 1.70 +/- 0.06). Saturating concentrations of fructose 1,6-bisphosphate (50 microM) activated PK-anoxic resulting in an enzyme with properties similar to fructose-1,6-bisphosphate-activated PK-aerobic, with K0.5 values for phosphoenolpyruvate of about 0.04 mM and Hill coefficients of 1.1. PK-anoxic showed much stronger regulation by the allosteric inhibitors MgATP, phenylalanine, proline and alanine. Fructose 1,6-bisphosphate partially relieved the inhibitions by ADP, MgATP, alanine, proline and arginine phosphate of both enzyme forms. However, at 0.1 mM phosphoenolpyruvate PK-aerobic was much more sensitive to activation by fructose 1,6-bisphosphate, Ka values being 0.05 +/- 0.01 microM for PK-aerobic and 1.3 +/- 0.1 microM for PK-anoxic. In the presence of 1.0 mM alanine and 1.5 mM MgATP much higher concentrations of fructose 1,6-bisphosphate were required for activation of PK-anoxic (Ka = 5.2 +/- 0.4 microM) than for PK-aerobic (Ka = 0.02 +/- 0.01 microM). Variations in pH over the range likely occurring in vivo during anaerobiosis caused no significant additional kinetic differences between the two enzyme forms. The dissimilarity in kinetic properties of PK-aerobic and PK-anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.