NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

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(corresponding author)

Abstract

Submitochondrial particles (SMP) were isolated from potato (Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O2, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O2 as acceptor) with optimum activity at pH 8 and a very low Km(NADH) of 3 μM. It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high Km(NADPH) of more than 1 mM. This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher Km(NADH) of 14 μM. It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low Km(NADPH) of 25 μM. This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.

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