Phosphorylation of Soybean (Glycine max L.) Nodule Phosphoenolpyruvate Carboxylase in Vitro Decreases Sensitivity to Inhibition by L-Malate

摘要: Phosphoenolpyruvate carboxylase (PEPC) from soybean (Glycine max L.Merr.) nodules was purified 187-fold to a final specific activity of 56 units mg-1 protein. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed one major polypeptide band, with molecular mass 110 kD, after the purification step. Two-dimensional PAGE resolved four isoelectric forms enzyme. Antibodies raised against enzyme immunoprecipitated PEPC desalted nodule extract. Two cross-reacting bands were obtained when protein immunoblots crude extracts subjected SDS-PAGE probed antiserum. One these corresponded 110-kD subunit PEPC, and other had about 60 kD. shown be activated in time-dependent manner preincubated Mg.ATP vitro. Activation observed assayed at pH 7 absence glycerol but not 8 presence glycerol. When o.5 mM L-malate included assay, activation much more pronounced than without malate. Maximal 30% 200% its presence. The concentrations producing 50% inhibition o.35 1.24 mM, respectively, before preincubation Mg.ATP. antiserum used immunoprecipitate extract that been Mg.[[gamma]-32P]ATP. then SDS-PAGE, followed by autoradiography. autoradiograph intense labeling following [[gamma]-32P]ATP. data suggest becomes phosphorylated an endogenous kinase, resulting decreased sensitivity results are discussed relation proposed functions legume nodules.