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  • Title: Purification and properties of four 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.
    Author: Rivoal J, Dunford R, Plaxton WC, Turpin DH.
    Journal: Arch Biochem Biophys; 1996 Aug 01; 332(1):47-57. PubMed ID: 8806708.
    Abstract:
    Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green alga Selenastrum minutum. PEPC1 is a homotetramer with a subunit M(r) of 102 kDa. PEPC2, PEPC3, and PEPC4 have respective native M(r)S of approximately 984, 1186, and 1590 kDa. SDS/PAGE analysis revealed that the latter three isoforms contain polypeptides having M(r)S of 102, 73, 70, 65, and 61 kDa. Immunoblot analyses and CNBr cleavage patterns suggest that the 102-kDa polypeptide present in all four isoforms is the same PEPC catalytic subunit. Our data suggest that the three high M(r)S PEPC isoforms are heteromeric protein complexes consisting of the 102-kDa PEPC1 catalytic subunit and immunologically unrelated polypeptides. Attempts to measure other enzyme activities associated with the protein complexes gave negative results. However, PEPC1 had immunological, physical, and kinetic properties very different from those of the larger M(r) PEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunoprecipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipitating 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 Mr PEPCs had greater apparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PEPC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate and malate). We conclude that the interaction of the PEPC catalytic subunit with unrelated polypeptides is responsible for the observed differences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activity in vivo.
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