PURIFICATION AND PROPERTIES OF 4 PHOSPHOENOLPYRUVATE CARBOXYLASE ISOFORMS FROM THE GREEN-ALGA SELENASTRUM-MINUTUM - EVIDENCE THAT ASSOCIATION OF THE 102-KDA CATALYTIC SUBUNIT WITH UNRELATED POLYPEPTIDES MAY MODIFY THE PHYSICAL AND KINETIC-PROPERTIES OF THE ENZYME

Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green alga Selenastrum minutum. PE PC1 is a homotetramer with a subunit M(r) of 102 kDa. PEPC2, PEPC3, an d PEPC4 have respective native M(r)s of approximately 984, 1186, and 1 590 kDa. SDS/PAGE analysis revealed that the latter three isoforms con tain polypeptides having M(r)s of 102, 73, 70, 65, and 61 kDa. Immunob lot analyses and CNBr cleavage patterns suggest that the 102-kDa poly- peptide present in all four isoforms is the same PEPC catalytic subuni t. Our data suggest that the three high M(r)s PEPC isoforms are hetero meric protein complexes consisting of the 102-kDa PEPC1 catalytic subu nit and immunologically unrelated polypeptides. Attempts to measure ot her enzyme activities associated with the protein complexes gave negat ive results. However, PEPC1 had immunological, physical, and kinetic p roperties very different from those of the larger M(r) PEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunop recipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipi tating activity toward PEPC1; (iii) PEPC1 was more heat sensitive than the other three isoforms; (iv) PEPC1 had a pH optimum of 9 versus 8.5 for the PEPC protein complexes; (v) the high M(r) PEPCs had greater a pparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PE PC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate a nd malate). We conclude that the interaction of the PEPC catalytic sub unit with unrelated polypeptides is responsible for the observed diffe rences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activity in vivo. (C) 19 96 Academic Press, Inc.