Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria

Eur J Biochem. 1991 Dec 5;202(2):617-23. doi: 10.1111/j.1432-1033.1991.tb16415.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism*
  • Cations, Divalent
  • Cell Membrane / physiology
  • Cell Membrane Permeability
  • Dicumarol / pharmacology
  • Hydrogen-Ion Concentration
  • Intracellular Membranes / physiology
  • Kinetics
  • Membrane Potentials
  • Mersalyl / pharmacology
  • Mitochondria / enzymology*
  • NADP / metabolism
  • NADPH Dehydrogenase / antagonists & inhibitors
  • NADPH Dehydrogenase / metabolism*
  • Oxidation-Reduction
  • Rotenone / pharmacology
  • Solanum tuberosum / enzymology*
  • Submitochondrial Particles / enzymology

Substances

  • Cations, Divalent
  • Rotenone
  • NADP
  • Mersalyl
  • Dicumarol
  • NADPH Dehydrogenase
  • Calcium