Characterization of NAD-dependent malate dehydrogenases from spinach leaves |
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Authors: | T Cvetić S Veljović-Jovanović Ž Vučinić |
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Institution: | (1) Faculty of Biology, University of Belgrade, Belgrade;(2) Center for Multidisciplinary Studies, University of Belgrade, Belgrade |
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Abstract: | Summary. 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 K
m 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.
Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska
43, 11000 Belgrade, Serbia. |
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Keywords: | : NAD-dependent malate dehydrogenase Spinacia oleracea Plasma membrane Chloroplast envelope membrane |
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