title: Characterization of NAD-dependent malate dehydrogenases from spinach leaves.
abstract: Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different Km values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools.
authors.affiliation: Institute of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia. tcvetic@bfbot.bg.ac.yu
authors.forename: T
authors.initials: T
authors.lastname: Cvetić
authors.forename: S
authors.initials: S
authors.lastname: Veljović-Jovanović
authors.forename: Z
authors.initials: Z
authors.lastname: Vucinić
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009243:NAD; D018515:Plant Leaves / Q000201:enzymology*; D018724:Spinacia oleracea / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D015415:Biomarkers; D007527:Isoenzymes; D009243:NAD; D008291:Malate Dehydrogenase