Biochemical and molecular characterization of high and low molecular mass isoforms of phosphoenolpyruvate carboxylase from developing castor oilseeds

Phosphoenolpyruvate carboxylase (PEPC) is a highly regulated and ubiquitous plant cytosolic enzyme that plays a central role in primary carbon metabolism. Recent purification of PEPC from developing castor oil seed (COS) endosperm isolated two PEPC isoforms. The association of a 107 kDa PEPC subunit (p107) with an immunologically-unrelated 64 kDa "bacterial-type" PEPC (BTPC) polypeptide (p64) leads to marked physical and kinetic differences between the native 410 kDa PEPC1 p107 homotetramer and novel 680 kDa PEPC2 p107/p64 hetero-octamer. Both p107 and a 118 kDa BTPC (p118) are readily proteolyzed in vitro (to p98 and p64, respectively) during COS PEPC purification. Prevention of the in vitro proteolysis of each subunit is vital for elucidation of the structural and regulatory characteristics of the COS PEPC2 complex and its hypothesized role in supporting fatty acid synthesis within developing COS. The creation of specific antibodies against COS p107 and BTPC has allowed the use of immunoblotting to characterize the in vitro degradation of both subunits by endogenous cysteine endopeptidase activity. COS p107 and p118 show greater susceptibility to proteolysis in extracts at acidic pH and later stages of COS development, which is prevented by a limited array of class-specific protease inhibitors. The specific in vitro N-terminal truncation of COS p107 to p98 by an asparaginyl endopeptidase was prevented by 2 mM 2,2'-dipyridyldisuIfide, an active site inhibitor of plant cysteine proteases. Immunological analysis of the COS BTPC confirmed its in vitro proteolysis from p118 to p64 by a different cysteine endopeptidase. PAGE of non-degraded vs. proteolyzed PEPC2 on calibrated non-denaturing gels followed by in-gel PEPC activity staining and immunoblotting indicated that: (i) native PEPC2 exists as a 910 kDa p118/p107 hetero-octamer, and (ii) p118 BTPC may function solely as a regulatory subunit within the PEPC2 complex. Identification and sequencing of cDNAs encoding COS p118 and p107 confirmed p118's characterization as a BTPC and its site of proteolysis, and provides valuable tools for future studies of the structure-function and physiological role of the novel PEPC2 hetero-oligomer in developing COS.