Phosphorylation of soybean (Glycine max L.) nodule phosphoenolpyruvate carboxylase in vitro decreases sensitivity to inhibition by L-malate

Phosphoenolpyruvate carboxylase (PEPC) from soybean (Glycine max L. Merr.) nodules was purified 187-fold to a final specific activity of 56 units mg^-1 of protein. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed one major polypeptide band, with a molecular mass of 110 kD, after the final purification step. Two-dimensional PAGE resolved four isoelectric forms of the purified enzyme. Antibodies raised against the purified enzyme immunoprecipitated PEPC activity from a desalted nodule extract. Two cross-reacting bands were obtained when protein immunoblots of crude nodule extracts subjected to SDS-PAGE were probed with the antiserum. One of these corresponded to the 110-kD subunit of PEPC, and the other had a molecular mass of about 60 kD. PEPC was shown to be activated in a time-dependent manner when desalted soybean nodule extracts were preincubated with Mg-ATP in vitro. Activation was observed when PEPC was assayed at pH 7 in the absence of glycerol but not at pH 8 in the presence of glycerol. When 0.5 mM L-malate was included in the assay, activation was much more pronounced than without malate. Maximal activation was 30% in the absence of L-malate and 200% in its presence. The L-malate concentrations producing 50% inhibition of PEPC activity were 0.35 and 1.24 mM, respectively, before and after preincubation with Mg-ATP. The antiserum against soybean nodule PEPC was used to immunoprecipitate PEPC from a desalted nodule extract that had been preincubated with Mg [γ-³²P]ATP. The immunoprecipitate was then subjected to SDS-PAGE, followed by autoradiography. The autoradiograph revealed intense labeling of the 110-kD subunit of PEPC following preincubation with [γ-³²P]ATP. The data suggest that soybean nodule PEPC becomes phosphorylated by an endogenous protein kinase, resulting in decreased sensitivity of the enzyme to inhibition by L-malate in vitro. The results are discussed in relation to the proposed functions of PEPC in legume nodules.