Abstract
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 o.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 o.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.[[gamma]-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 [[gamma]-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.
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- Anderson M. P., Heichel G. H., Vance C. P. Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules. Plant Physiol. 1987 Sep;85(1):283–289. doi: 10.1104/pp.85.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christeller J. T., Laing W. A., Sutton W. D. Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development. Plant Physiol. 1977 Jul;60(1):47–50. doi: 10.1104/pp.60.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coker G. T., Schubert K. R. Carbon Dioxide Fixation in Soybean Roots and Nodules: I. CHARACTERIZATION AND COMPARISON WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING EARLY NODULE DEVELOPMENT. Plant Physiol. 1981 Apr;67(4):691–696. doi: 10.1104/pp.67.4.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doucet J. P., Trifaró J. M. A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution. Anal Biochem. 1988 Feb 1;168(2):265–271. doi: 10.1016/0003-2697(88)90317-x. [DOI] [PubMed] [Google Scholar]
- Jiao J. A., Chollet R. Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants. Plant Physiol. 1991 Apr;95(4):981–985. doi: 10.1104/pp.95.4.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiao J. A., Vidal J., Echevarría C., Chollet R. In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum. Plant Physiol. 1991 May;96(1):297–301. doi: 10.1104/pp.96.1.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Job D., Cochet C., Dhien A., Chambaz E. M. A rapid method for screening inhibitor effects: determination of I50 and its standard deviation. Anal Biochem. 1978 Jan;84(1):68–77. doi: 10.1016/0003-2697(78)90484-0. [DOI] [PubMed] [Google Scholar]
- King B. J., Layzell D. B., Canvin D. T. The role of dark carbon dioxide fixation in root nodules of soybean. Plant Physiol. 1986 May;81(1):200–205. doi: 10.1104/pp.81.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laing W. A., Christeller J. T., Sutton W. D. Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation. Plant Physiol. 1979 Mar;63(3):450–454. doi: 10.1104/pp.63.3.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pathirana S. M., Vance C. P., Miller S. S., Gantt J. S. Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules. Plant Mol Biol. 1992 Nov;20(3):437–450. doi: 10.1007/BF00040603. [DOI] [PubMed] [Google Scholar]
- Plaxton W. C. Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf. Eur J Biochem. 1989 May 1;181(2):443–451. doi: 10.1111/j.1432-1033.1989.tb14745.x. [DOI] [PubMed] [Google Scholar]
- Schuller K. A., Turpin D. H., Plaxton W. C. Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase. Plant Physiol. 1990 Nov;94(3):1429–1435. doi: 10.1104/pp.94.3.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Snapp S. S., Vance C. P. Asparagine Biosynthesis in Alfalfa (Medicago sativa L.) Root Nodules. Plant Physiol. 1986 Oct;82(2):390–395. doi: 10.1104/pp.82.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vance C. P., Stade S. Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase. Plant Physiol. 1984 May;75(1):261–264. doi: 10.1104/pp.75.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vance C. P., Stade S., Maxwell C. A. Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids. Plant Physiol. 1983 Jun;72(2):469–473. doi: 10.1104/pp.72.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weaver C. D., Crombie B., Stacey G., Roberts D. M. Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26. Plant Physiol. 1991 Jan;95(1):222–227. doi: 10.1104/pp.95.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]