Analysis of biophysical differences between oxidized and reduced chloroplast NADP-malate dehydrogenase.
Various properties of purified chloroplast NADP-malate dehydrogenase were analyzed with respect to the redox state of the light/dark-modulated enzyme. The reduced enzyme is less resistant to heat, but the instability can be overcome by the addition of the coenzyme NADPH. Similarly, instability of the reduced NADP-MDH at high pH is alleviated by NADPH. The kinetics and protection characteristics of the alkylation of accessible thiols of NADP-MDH are used to describe the location of essential thiols relative to the active site, since again the coenzyme protects the active enzyme very effectively from inactivation by alkylation. The increased hydrophobicity of the reduced as opposed to the oxidized enzyme becomes apparent as the loss of activity from solutions due to adsorption to plastic surfaces. The kinetics and the solvent dependency of this process are analyzed and discussed, both from the practical (recovery of the purified enzyme) and the physiological point of view (in vivo protein/protein and protein/membrane interactions). The oxidized NADP-MDH has a lower tendency to bind to solid surfaces. NADP(H) efficiently prevents adsorption of the reduced form. Macromolecular solvents (polyethylene glycol), detergents (Triton X-100), or competing proteins also protect this otherwise very hydrophobic form from irreversible loss due to adsorption. Ribulosebisphosphate carboxylase/oxygenase and polyethylene glycol 10,000, however, used as competing substances only keep the oxidized, not the reduced, NADP-MDH in solution.
Scheibe
R
R
Pflanzenphysiologie, Universität Osnabrück, Germany.
Geissler
A
A
Rother
T
T
D002736:Chloroplasts / Q000201:enzymology*; D003624:Darkness; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology; D007700:Kinetics; D008027:Light; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011092:Polyethylene Glycols / Q000494:pharmacology; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics
D011092:Polyethylene Glycols; D009249:NADP; D008291:Malate Dehydrogenase; D012273:Ribulose-Bisphosphate Carboxylase; D005033:Ethylmaleimide