Structural, immunological and kinetic comparisons of NADP‐dependent malate dehydrogenases from spinach (C₃) and corn (C₄) chloroplasts

Abstract

This paper compares structural, immunological and kinetic properties of corn (C,) and spinach (C,) NADPmalate dehydrogenases. These chloroplastic enzymes are regulated in vivo by thiol -disulfide interchange. Both in their oxidized (inactive) and reduced (active) states these enzymes have a dimeric structure with molecular masses for the subunit ranging from 28 kDa to 38 kDa according to the procedure used for the determination. These enzymes are thus structurally related. The use of specific antibodies showed that they are also immunologically related although not identical. Finally both enzymes showed close kinetic properties with comparable k,,, and K,. Since C, plants have approximately ten times more NADP-malate dehydrogenase activity than C3 plants, these data suggest that the differences in activities are probably related to the enzyme content of each plant type.N ADH-dependent malate dehydrogenases are ubiquitous enzymes, isoenzymes of which are found in bacteria, mammals and plants [l]. The latter organisms, however, possess a unique NADPH-dependent malate dehydrogenase (NADP-MDH). The most striking difference between the NADH and the NADPH-dependent malate dehydrogenases is that the latter, unlike its NADH counterparts, is regulated by its redox status [2, 31. Indeed, the NADP-malate dehydrogenase is in vivo activated by light and inactivated in the dark [4, 51. Studies of the activation/inactivation mechanism have led to the discovery of a light-dependent modification system, endogeneous to the chloroplast, called the ferredoxin-thioredoxin system, which can be reconstituted in vitro and comprises isolated thylakoids and the following soluble proteins: ferredoxin, ferredoxin-thioredoxin reductase and thioredoxins [6, 71. Photochemically or chemically (dithiothreitol) reduced thioredoxins can, in turn, activate the NADP-malate dehydrogenase presumably via a thiol -disulfide interchange mechanism with the appearance of new thiol groups on the enzyme together with the catalytic activity [8, 91. NADP-MDH can also be reduced by dithiothreitol alone with a lower efficiency through a similar mechanism [lo-121.NADP-malate dehydrogenase is present only in the chloroplasts of C3 and C4 mesophyll plant cells [13,14]. While it seems to be clear that in C4 leaves it is involved in the soCorrespvndence to J

The Ferredoxin/Thioredoxin System of Oxygenic Photosynthesis

“…In land plants, the activity of NADP-MDH is strictly controlled by reversible reduction of the two redox active disulfide bridges through the Fdx/Trx system. C4 plants which trap CO 2 as malate contain a significantly higher level of NADP-MDH than their C3 counterparts (71). Although its mechanism of redox regulation is rather complex, NADP-MDH is the best understood of the light-activated enzymes of chloroplasts.…”

Activation of NADP-malate dehydrogenase in C3 plants by reduced glutathione

“…They suggested that two disulphide bonds were reduced in vitro during the activation of the maize enzyme compared with 1 disulphide with the pea enzyme. Ferte et al (1986) reported similar but not identical amino acid composition between the maize and pea enzyme; however they reported that both enzymes had three cysteine per monomer.…”

Amino Acid Sequence and Molecular Weight of Native NADP Malate Dehydrogenase from the C4 Plant Zea mays

“…More recent studies report higher subunit mol wts of between 42,000 and 43,000 (7,10). Some workers find the native forms of both the active (reduced, light activated) and unactivated (oxidized, dark inactivated) forms ofNADP-MDH (4) to be apparent dimers with a mol wt of about 88,000 (5,10). However, other studies using gel filtration procedures have indicated higher mol wts (2) and that the activated enzyme has a higher apparent mol wt than the unactivated enzyme (1 1).…”