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 (R icinus communis L.) has been purl fled 3900-fold to apparent homogenei ty and a final specific activity of 51 mu mol of pyruvate produced/min /mg protein; PAGE, immunoblot, and gel filtration analyses off the fin al preparation indicated that this enzyme is an alpha(2) beta(2) heter otetramer of about 250 kDa that is composed of an equivalent ratio of 57- sind 56-KDa subunits. The enzyme was relatively heat-stable and di splayed a broad pH optimum of approximately 6.5. However, optimal effi ciency in substrate utilization [in terms of V-max/K-m for phosphoenol pyruvate (PEP) or ADP] occurred at pH 7.5, Enzyme activity was absolut ely dependent upon the simultaneous presence of bivalent and a univale nt metal cation, with Mg2+ and K+ fulfilling this:requirement. Hyperbo lic saturation kinetics mere observed with PEP, ADP, and K+, whereas M g2+ binding exhibited positive cooperativity. Mg, citrate, oxalate, an d glutamate were the most effective inhibitors at pH 7.5. inhibition b y these compounds was more pronounced at pH 7.5 than at pH 6.5 and the y yielded additive inhibition when tested in pairs. Aspartate function ed as an activator by facilitating the binding of PEP and relieving th e inhibition of PK, by glutamate. The in vivo activity of leaf PK,is p robably regulated by the relative cytosolic levels of citrate, glutama te, and aspartate. This provides a possible rationale for the known ac tivation of leaf PK, that occurs during periods of enhanced ammonia as similation. Together with our previous studies, the results also indic ate that castor oil plant PK, exists as tissue-specific isoforms that demonstrate substantial differences in their respective physical and/o r kinetic and regulatory properties. (C) 1996 Academic Press, Inc.