title: Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.
abstract: Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.
authors.affiliation: Department of Plant Physiology, University of Lund, Sweden.
authors.forename: A G
authors.initials: AG
authors.lastname: Rasmusson
authors.forename: I M
authors.initials: IM
authors.lastname: Møller
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability; D001728:Dicumarol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials; D008634:Mersalyl / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D012402:Rotenone / Q000494:pharmacology; D011198:Solanum tuberosum / Q000201:enzymology*; D013367:Submitochondrial Particles / Q000201:enzymology
chemical_list: D002413:Cations, Divalent; D012402:Rotenone; D009249:NADP; D008634:Mersalyl; D001728:Dicumarol; D009252:NADPH Dehydrogenase; D002118:Calcium