Journal
PLANT SCIENCE
Volume 151, Issue 1, Pages 29-37Publisher
ELSEVIER SCI IRELAND LTD
DOI: 10.1016/S0168-9452(99)00191-0
Keywords
Sorghum; seed phosphoenolpyruvate carboxylase; enzyme kinetics; protein phosphorylation; salt stress
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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 polypeptides (108 and 110 kDa) were resolved by SDS/PAGE and shown to increase, 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 imbibing 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 phosphorylation were severely inhibited in the presence of NaCl in the imbibing medium, although PEPCk content was not altered. However, in vitro, NaCl had no effect on both PEPCk activity and PEPC phosphorylation. On the other hand, L-malate was a potent inhibitor of seed PEPCk activity in in vitro assays. Since NaCl also decreased the rate of L-malate consumption in the imbibing grain, the salt inhibition of PEPC phosphorylation was suggested to be due to the concentration-dependent blocking of PEPCk activity in vivo by this compound. Consistent with these data, germination and PEPC phosphorylation were inhibited, while PEPCk levels were not altered, when seeds were germinated in the presence of L-malate. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
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