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Protoplasma

, Volume 232, Issue 3–4, pp 247–253 | Cite as

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

  • T. Cvetić
  • S. Veljović-Jovanović
  • Ž. Vučinić
Article

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.

Keywords: NAD-dependent malate dehydrogenase; Spinacia oleracea; Plasma membrane; Chloroplast envelope membrane. 

Abbreviations:

MDH

malate dehydrogenase

NAD-MDH

NAD-dependent malate dehydrogenase

HPR

hydroxypyruvate reductase

IEF

isoelectric focusing

OAA

oxaloacetate

ADH

alcohol dehydrogenase

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • T. Cvetić
    • 1
  • S. Veljović-Jovanović
    • 2
  • Ž. Vučinić
    • 2
  1. 1.Faculty of BiologyUniversity of BelgradeBelgrade
  2. 2.Center for Multidisciplinary StudiesUniversity of BelgradeBelgrade

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