Effect of Lipids on a Membrane-Bound NADH Dehydrogenase from Bacillus caldotenax

Abstract

NADH dehydrogenase [EC 1.6.99.3] in membranes of Bacillus caldotenax was solubilized with sodium N-lauroylsarcosinate and purified 50-fold from membranes to 75–80% homogeneity, as judged by SDS-polyacrylamide gel electrophoresis. The enzyme was considered to be located on the electron transport chain and to be an FAD-containing protein. The molecular weight of the subunit was estimated to be 44, 000. The enzyme (or the enzyme bound to the B. caldotenax membrane lipids) follows a ping-pong mechanism. The enzyme can oxidize NADH, but not NADPH, with 2, 6-dichlorophenol indophenol, ferricyanide, menadione, and cytochrome C as electron acceptors. Membrane lipids or Triton X-100 stimulated the enzyme activity, except that with menadione. Lipids decreased the apparent affinity of electron acceptors and NADH to the enzyme, and increased the maximum velocity, except when menadione was used as the electron acceptor. Lipids partially protected the enzyme from thermal inactivation. The enzyme exhibited a continuous Arrhenius plot, while the lipids- or membrane-bound enzyme exhibited a discontinuous plot.