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Carbon metabolism in germinating Ricinus communis cotyledons. Purification, characterization and developmental profile of NADP‐dependent malic enzyme
Author(s) -
Colombo S. L.,
Andreo C. S.,
Podestá F. E.
Publication year - 1997
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1997.tb01069.x
Subject(s) - biochemistry , malic enzyme , biology , enzyme , citric acid cycle , oxidoreductase , citrate synthase , cofactor , enzyme assay , fatty acid , dehydrogenase
The possible implication of NADP‐dependent malic enzyme (NADP‐ME; L‐malate:NADP oxidoreductase [oxaloacetate‐decarboxylating], EC 1.1.1.40) in fatty acid synthesis was examined in Ricinus communis L. cotyledons, NADP‐ME catalyses the conversion of L‐malate to pyruvate and NADPH, potential substrates for fatty acid synthesis. NADP‐ME activity and protein levels were monitored during germination, up to 20 days postimbibition. The developmental profile showed a peak in activity (6 times with respect to the basal value) and immunoreactive protein (a single 72‐kDa band using anti‐maize NADP‐ME antibodies) around day 7. The enzyme was partially purified (41‐fold) and its kinetics characterized. The optimum pH was around 7.1. K m values for L‐malate and NADP + were 0.68 m M and 8.2 μ M respectively. The enzyme used Mg 2+ or Mn 2+ as essential cofactors. Several metabolites were assayed as potential enzyme modulators. Succinate, CoA, acetyl‐CoA and palmitoyl‐CoA were activators of NADP‐ME, at saturating or sub‐saturating substrate concentrations, K 2 values for CoA and derivative compounds were in the micromolar range (i.e., 0.8 μ M for acetyl‐CoA). No significant effects were obtained with other Krebs cycle intermediates and amino acids (i.e. 2‐oxoglutarate, glutamate, glutamine, fumarate). The activity was 29 times higher in the forward (decarboxylating) direction compared to the reverse direction. These results hint at cotyledon NADP‐ME behaving as a regulatory enzyme in R. communis . Its activity is responsive to metabolites of the fatty acid synthesis pathway, and thus a role in this metabolism is suggested.