Purification and characterization of cytosolic pyruvate kinase from leaves of the castor oil plant

Cytosolic pyruvate kinase (PKc) from leaves of the castor oil plant (Ricinus communis L.) has been purl fled 3900-fold to apparent homogeneity and a final specific activity of 51 mu mol of pyruvate produced/min/mg protein; PAGE, immunoblot, and gel filtration analyses off the final preparation indicated that this enzyme is an alpha(2) beta(2) heterotetramer of about 250 kDa that is composed of an equivalent ratio of 57- sind 56-KDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of approximately 6.5. However, optimal efficiency in substrate utilization [in terms of V-max/K-m for phosphoenolpyruvate (PEP) or ADP] occurred at pH 7.5, Enzyme activity was absolutely dependent upon the simultaneous presence of bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this:requirement. Hyperbolic saturation kinetics mere observed with PEP, ADP, and K+, whereas Mg2+ binding exhibited positive cooperativity. Mg, citrate, oxalate, and glutamate were the most effective inhibitors at pH 7.5. inhibition by these compounds was more pronounced at pH 7.5 than at pH 6.5 and they yielded additive inhibition when tested in pairs. Aspartate functioned as an activator by facilitating the binding of PEP and relieving the inhibition of PK, by glutamate. The in vivo activity of leaf PK,is probably regulated by the relative cytosolic levels of citrate, glutamate, and aspartate. This provides a possible rationale for the known activation of leaf PK, that occurs during periods of enhanced ammonia assimilation. Together with our previous studies, the results also indicate that castor oil plant PK, exists as tissue-specific isoforms that demonstrate substantial differences in their respective physical and/or kinetic and regulatory properties. (C) 1996 Academic Press, Inc.