Posttranslational regulation of phosphoenolpyruvate carboxylase during germination of Sorghum seeds: influence of NaCl and L-malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31: PEPC) was characterized in de -embryonated Sorghum seeds, focusing on the interaction between metabolites and posttranslational control of the enzyme by phosphorylation. Two PEPC p olypeptides (108 and 110 kDa) were resolved by SDS/PAGE and shown to increa se, in parallel with PEPC activity during seed germination. PEPC displayed very low K-m values for PEP (90 mu M) and inhibition constant (IC50) for L- malate (75 mu M) in desalted protein extracts from de-embryonated dry seeds . The inhibition of PEPC by 0.16 mM L-malate, pH 7.3, decreased from 70 to 30%, along with a consistent increase in IC50 (75-220 mu M) after 5 days of germination. PEPC phosphorylation was established both in vivo, after imbi bing the seeds with [P-32]phosphate, and in vitro in reconstituted assays. A PEPC kinase (PEPCk) was partially purified from seed protein extracts by blue dextran agarose chromatography and shown to be independent of calcium and to phosphorylate both seed and recombinant C-4 PEPC from Sorghum on the enzyme's N-terminal domain. Seed germination, PEPC accumulation and phosph orylation were severely inhibited in the presence of NaCl in the imbibing m edium, although PEPCk content was not altered. However, in vitro, NaCl had no effect on both PEPCk activity and PEPC phosphorylation. On the other han d, L-malate was a potent inhibitor of seed PEPCk activity in in vitro assay s. Since NaCl also decreased the rate of L-malate consumption in the imbibi ng grain, the salt inhibition of PEPC phosphorylation was suggested to be d ue to the concentration-dependent blocking of PEPCk activity in vivo by thi s compound. Consistent with these data, germination and PEPC phosphorylatio n were inhibited, while PEPCk levels were not altered, when seeds were germ inated in the presence of L-malate. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.