Nathalie Ferté

Molecular properties and thioredoxin-mediated activation of spinach chloroplastic NADP-malate dehydrogenase

There is no doubt that light-generated reducing equivalents can be used in C3 plants to perform a number of reactions occurring outside the chloroplasts [1-3]. Since the chloroplastic membrane is impermeable to NADPH and to reduced ferredoxin, it is currently believed that these reduction reactions in the cytoplasm involve the so-called C4dicarboxylic acid shuttle [4]. A specific malate translocator located on the chloroplastic membrane exports, outside the chloroplast, this dicarboxylic acid which is used in the cytosol to perform a number of reduction reactions [3]. NADPH formed during photosynthesis is used to reduce oxaloacetate to malate in the chloroplastic stroma. Chloroplastic malate dehydrogenase plays therefore an important role in the generation of reducing power used in reactions occurring in the cytoplasm. Moreover, crude extracts containing chloroplastic malate dehydrogenase are totally inactive in the dark and become active upon illumination. The effect of light is mimicked by a mixture of dithiothreitol and thioredoxin [5,6]. Surprisingly, chloroplastic NADP-malate dehydrogenase has never been purified to homogeneity. Therefore, nothing is known as to its structure, its reaction mechanism and the molecular bases of photoregulation by thioredoxin. Moreover one totally ignores whether several iso-forms of malate dehydrogenase exist in the chloroplast. The aim of this paper is to describe a purification procedure of chloroplastic NADP-malate dehydrogenase, to report some molecular properties of this enzyme as well as some features of its activation by dithiothreitol and thioredoxin.

Control of Chloroplastic NADP-malate dehydrogenase activity by thioredoxins

Abstract Oxidized (inactive) chloroplastic NADP-malate dehydrogenase is 56-k Da homodimer, the two subunits of which are bound by a disulfide bridge. The enzyme is activated by three distinct chloroplast thioredoxins, thioredoxin m , f A and f B . When reduced by these proteins, the active enzyme is stable, whereas when reduced by dithiothreitol, activity is unstable. This result is apparently the consequence of aggregation of the reduced protein during prolonged incubation with dithiothreitol. At pH-values similar to those in the chloroplast stroma in the dark (pH 7), the reduced enzyme is still active.

Purification of several NADP-dependent malate dehydrogenase isozymes from spinach leaves. Kinetic properties

Abstract Four NADP-malate dehydrogenase (NADP-MDH) (EC 1.1.1.82) isozymes from spinach leaves have been purified to apparent homogeneity by DEAE-cellulose and affinity chromatography, and molecular sieving. They have the same native molecular weight (≈57 000) as determined by sedimentation equilibration analysis. They can be distinguished by their eletrocphoretic and chromatographic (over DEAE-cellulose) behaviors. On this basis, they constitute two sets of two enzymes each: MDH-A and -B, and MDH-C and -D. Catalytic properties of the most abundant isozyme, MDH-A, have been studied. Both NADPH and oxaloacetate inhibit the forward reductive reaction. Other kinetic parameters are also presented.

Study of Kinetic and Binding Properties of NADP-malate Dehydrogenase (MDH a) from Spinach Chloroplasts

We have obtained homogeneous the four isoenzymes of NADP-MDH. One of them, MDH A, has been studied with details. We present here the results of a steady state kinetic study including effects of the products. For checking the proposed reactional mechanism, we have performed an equilibrium binding study with substrates and products. NADP-MDH is an enzyme which plays a role in the regulation of the export of reducing power from stroma chloroplast to cytosol (N. Ferte et al., 1982).

Regulation of the Export of the Reducing Power from Chloroplast to Cytosol in Relation with Molecular Properties of Chloroplastic NADP-MDH

We have already described the purification of a chloroplastic NADP-malate dehydrogenase (Ferte et al., 1982). It is a 56 000 daltons dimeric protein made up of two apparently identical subunits. This enzyme plays an important role in the generation of reducing power used in the reduction cytoplasmic reactions. NADPH from photosynthesis reduces oxaloacetate to malate in the stroma. This reaction is catalyzed by NADP-MDH. A malate translocator exports this acid outside the chloroplast where NAD-MDH generates NADH.