Effect of lipids on a membrane-bound NADH dehydrogenase from Bacillus caldotenax

NADH dehydrogenase [EC 1.6.99.3] in membranes of Bacillus caldotenax was solu-bilized with sodium TV-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 activ-ity, 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. Many investigations on the physiology, and espe-cially on the thermal properties of cytoplasmic enzymes, of thermophilic bacteria have been re-ported (/). In contrast to the cytoplasmic en-zymes, few reports on the membrane-bound or respiratory chain-linked enzymes of thermophilic bacteria have been presented. Recently the ter-minal oxidoreductase, cytochrome c oxidase [EC 1.9.3.1], of the respiratory chain was highly puri-fied from Thermits thermophilus (2) and purified to homogeneity from a thermophilic bacterium PS2(5, Abbreviations: sarcosinate, sodium iV-lauroylsarcosi-nate; DC1P, 2,6-dichlorophenol indophenol; DTT