NADH dehydrogenase and NADH oxidation in membrane vesicle from Bacillus subtilis.

Abstract

Crossed immunoelectrophoresis of membrane vesicles of Bacillus subtilis W23, solubilized in 5% Triton X‐100, against anti‐vesicle imunoglobulins resulted in at least 43 immunoprecipitates.With the zymogram staining techique the precipitation lines of the enzymes succinic dehydrogenase, malate dehydrogenase, ATPase, NADPH dehydrogenase and NADH dehydrogenase were identified.NADH‐dehydrogenase activity was found in three precipitation lines. Preparative polyacrylamide gel electrophoresis of NADH dehydrogenase, solubilized with 0.1% Triton X‐100, showed three to five major protein bands with NADH dehydrogenase activity.The sodium dodecyl sulphate/polyacrylamide gel electrophoresis patterns of these bands all contained a band of the catalytic subunit with an apparent Mr of 64000. Antibodies against this catalytic subunit were prepared. The presence of only one NADH dehydrogenase in the cytoplasmic membrane was indicated by the facts that solubilized (NADH dehydrogenase) immunoglobulins and that membranes solubilized with 5% Triton X‐100 showed the same three precipitation lines with NADH dehydrogenase activity when either anti‐(NADH dehydrogenase) or anti‐(membrane vesicle) immunoglobulins were used.This NADH dehydrogenase is located at the inner surface of the cytoplasmic membrane(like succinic dehydrogenase and ATPase) as was shown with immunoabsorption experiments.Membrane vesicles oxidize NADH, presented at the outer surface, at a high rate. The mechanism of NADh oxidation was studied.NADH is oxidized via the respiratory chain. Uptake of NADH by the membrane vesicles could not be demonstrated and ermoval of 74% of NADH dehydrogenase resulted only in a 24% decrease of NADH oxidase activity. Membrane vesicles from the menaquinone‐deficient strain B subtilis aroD do not oxidize externally added NADH but BADH oxidation can be restord by addition of the menaquinone analogue, menadione. These resuts suggest that externally added NADH donates electrons directly to menaquinone‐7 at the outer surface of the vesicle membrane and that NADH dehydrogenase is not involved in this oxidation process.