Abstract
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32, PEPCK) from rainbow trout liver and carp hepatopancreas were purified and characterized kinetically. There are two forms of PEPCK in the rainbow trout liver, but only one form in the carp hepatopancreas. The molecular weight was 61, 000 for rainbow trout liver major isozyme and 59, 000 for carp hepatopancreas enzyme when determined by Sephadex G-100 gel filtration. However, when determined by SDS poly-acrylamide gel electrophoresis the former was 87, 000 and the latter was 83, 000. The optimum pH for both enzymes was 7.7 in the direction of oxalacetate (OAA) decarboxylation, and 7.2 in the direction of phosphoenolpyruvate (PEP) carboxylation. Enzyme activities depended on the concentration of substrates (OAA or PEP) and nucleotides in either direction. Both enzymes were also strongly inhibited by several nucleotides. Divalent cation was absolutly necessary for both enzymes in either direction, and Mn2+was found to be the best activator among several metal ions. Both enzymes were strongly inhibited by Zn2+, especially in the reaction of OAA decarboxylation. Co2+ and Cu2+ also showed obvious inhibition, but Ca2+ showed ambiguous effect on both enzymes. The kinetic properties of PEPCK from both fish liver were similiar not only qualitatively but also quantitatively.
