PURIFICATION AND CHARACTERIZATION OF A NOVEL PHOSPHOENOLPYRUVATE CARBOXYLASE FROM BANANA FRUIT

Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa caven dishii L.) fruits has been purified 127-fold to apparent homogeneity a nd a final specific activity of 32 mu mol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resol ved a single protein-staining band that co-migrated with PEPC activity . Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, whic h stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides ar e related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contras t to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha(2) beta(2) heterotetramer. Monospecific rab bit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established t hat the 100 kDa subunit did not arise via proteolysis of the 103 kDa s ubunit after tissue extraction, and that the subunit composition of ba nana PEPC remains uniform throughout the ripening process. PEPC displa yed a typical pH activity profile with an alkaline optimum and activit y rapidly decreasing below pH 7.0. Enzymic activity was absolutely dep endent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ f ulfilling this requirement. The response of the PEPC activity to PEP c oncentration and to various effecters was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-mo nophosphates and potently inhibited by malate, succinate, aspartate an d glutamate at pH 7.0, whereas the effect of these metabolites was con siderably diminished or completely abolished at pH 8.0. The significan ce of metabolite regulation of PEPC is discussed in relation to possib le functions of this enzyme in banana fruit metabolism.