Renate Scheibe

NADP regulates the light activation of NADP-dependent malate dehydrogenase.

The chloroplastic NADP-dependent malate-dehydrogenase (EC 1.1.1.82) activity is modulated by light and dark. The enzyme is activated upon illumination of intact or broken chloroplasts or by incubation with dithiothreitol, whereas dark has the opposite effect. The present communication shows an additional regulation of the light modulation: in isolated intact pea chloroplasts, light activation was inhibited in the presence of electron acceptors such as sodium bicarbonate, 3-phosphoglycerate or oxaloacetate, which consume NADPH2 and produce NADP. With broken chloroplasts, addition of NADP resulted in a pronounced lag phase of NADP-dependent malate dehydrogenase light activation, while NADPH2 was without any effect. The extent of the lag phase was correlated to the amount of NADP added. When light was replaced by dithiotreitol, the inhibition effect was even more pronounced. It was assumed that NADP inhibits the modulation reaction directly: reduced thioredoxin, a potent mediator of activation by light, or dithiotreitol appear to counteract NADP in a competitive manner. The results indicate a physiological role of NADP in the regulation of chloroplastic NADP-dependent malate dehydrogenase which is capable of removing electrons from the chloroplast, via oxaloacetate reduction and malate export. Thus an NADP concentration sufficient for continuous photosynthetic electron flow may be achieved.

Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves

A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer.

Thioredoxinm in pea chloroplasts: Concentration and redox state under light and dark conditions

Thioredoxins have been characterized as small, heat-stable proteins which have multiple functions as redox carriers in various bacterial, plant and mammalian systems [ 1 ]. For Escherichia coli thioredoxin a change in tile redox state due to the dithiol/disulfide group in the active center has been shown [2]. Thioredoxin can be reduced either by dithiothreitol (DTT) or enzymatically. In plants they are involved in the modulation of several enzymes [3]; reduced via photosynthetic electron flow by ferredoxin-thioredoxinreductase. When this flow stops, thioredoxin is oxidized by a yet unknown oxidant. Both the reduced and the oxidized form specifically mediate the activation or inactivation of chloroplast enzymes, as shown for NADP-dependent malate dehydrogenase and glucose 6-P-dehydrogenase [4]. Several thioredoxins in plant systems have been described which appear to differ in Mr-value and specificity with respect to the modulated enzymes [5,6]. Spinach chloroplasts contain 3 thioredoxin isomers with distinct isoelectric points [7]. The molecular mechanism of the modulation of chloroplast enzymes has not yet been elucidated. One possibility is a reduction of the enzymes as in the case of insulin, where thioredoxin acts as a catalytic hydrogen carrier [8]. Another mode of action is the formation of a stable complex with the enzyme as in a phage-induced DNA-polymerase ofE. coli [91. In the chloroplast system it is important to note that lightor dark-modulation apparently reaches completion in terms of seconds [ 10]. In a first attempt

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ' giant rosette' plants*

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.

Studies on the mechanism of the reductive activation of NADP-malate dehydrogenase by thioredoxin m and low-molecular-weight thiols

Chloroplast NADP-malate dehydrogenase ((S)-malate:NADP+ oxidoreductase, EC 1.1.1.82) purified from pea (Pisum sativum) leaves can be activated by dithiothreitol-reduced chloroplast thioredoxin m or by reduction with dithiothreitol alone. Both thioredoxin m-dependent and dithiothreitol-dependent activation of NADP-malate dehydrogenase were more rapid at higher ionic strength (up to 2–3 M sult). Thioredoxin m-catalyzed dithiothreitol-dependent activation was maximal at 20 mM dithiothreitol, whereas direct activation of NADP-malate dehydrogenase by dithiothreiol was not saturated at 300 mM dithiothreitol. Both dithiothreitol and thioredoxin m-catalyzed activation were more rapid at higher pH, presumably reflecting ionization of the thiol groups of the dithiothreitol or thioredoxin m. Although dithiothreitol was the best low-molecular-weight activating agent, various monothiols, including 2-mercaptoethanol and glutathione, were capable of activating NADP-malate dehydrogenase either directly or via a thioredoxin-catalyzed reaction. Implications of this observation for the interpretation of previous results are discussed.

Quantitation of the thiol groups involved in the reductive activation of NADP: Malate dehydrogenase

Abstract (1) The amino-acid analysis of homogeneous NADP: malate dehydrogenase ( l -malate: NADP+ oxidoreductase, E.C. 1.1.1.82) from pea leaves revealed the presence of three half-crystine residues per subunit Mr 38500). The determination of the total thiol groups of the denatured and reduced enzyme which was performed by incorporation of N-[ 3 H ] ethylmaleimide as well as by measuring the reaction with 5,5′ dithiobis(2-nitrobenzoic acid) spectrophotochemically were in full agreement with this finding. (2) It could be established that upon activation of NADP: malate dehydrogenase two thiol groups per subunit are formed. The third thiol is apparently not available in the native enzyme. The kinetics of thiol group formation and of expression of catalytic activity suggested that the fully reduced tetramer is the only species exhibiting NADP: malate dehydrogenase activity. (3) The inhibition of activation of NADP: malate dehydrogenase by NADP could be explained in terms of the bound NADP preventing the reduction of the regulatory disulfide bridge

Carbon dioxide assimilation and stomatal response of afroalpine giant rosette plants

Maximal rates of CO2 assimilation of 8–11 μmol m-2 s-1 at ambient CO2 concentration were measured for Dendrosenecio keniodendron, D. brassica, Lobelia telekii and L. keniensis during the day in the natural habitat of these plants at 4,200 m elevation on Mt. Kenya. Even at these maximal rates, the CO2 uptake of all species was found to correspond to the linear portion of the CO2 response curve, with a calculated stomatal limitation for CO2 diffusion of 42%. Photosynthesis was strongly reduced at temperatures above 15° C. In contrast to this sensitivity to high temperatures, frozen leaves regained full photosynthetic capacity immediately after thawing. Stomata responded to dry air, but not to low leaf water potentials which occurred in cold leaves and at high transpiration rates. During the day reduced rates of CO2 uptake were associated with reduced light interception due to the erect posture of the rosette leaves and with high temperatures. Stomata closed at vapour pressure deficits which were comparable in magnitude to those characteristic of many lowland habitats (40 mPa Pa-1).

Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers.

Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 M guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0-8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by oxaloacetate.

Use of tentoxin and nigericin to investigate the possible contribution of ΔpH to energy dissipation and the control of electron transport in spinach leaves

(a) Spinach leaf discs were floated overnight on various concentrations of tentoxin, an ATP synthase inhibitor, or nigericin, an uncoupler. They were then illuminated in saturating CO 2 . (b) With tentoxin, the inhibition of photosynthesis was accompanied by lower ATP/ADP ratios and increased ‘energy’ quenching of chlorophyll fluorescence. There was a small increase in the reduction of the Photosystem II acceptor, Q A , as monitored by photochemical quenching of chlorophyll fluorescence. However, activation of NADP-malate dehydrogenase decreased, showing that the acceptor side of Photosystem I becomes more oxidised, (c) With nigericin, the inhibition of photosynthesis was accompanied by decreased ATP/ADP ratios, decreased energy quenching, Q A became more reduced than with tentoxin, and the acceptor side of Photosystem I also became more reduced, (d) The results are used to discuss the contribution of “energy” quenching to energy dissipation, and the operation of photosynthetic control in leaves.