Purification and Properties of Malate-NAD+ Dehydrogenase of Moraxella lwoffi (N.C.I.B. 8250)

Abstract

Many strict aerobes have a particulate (non-NAD-linked) malate dehydrogenase (Jurtshuk et al., 1969), whereas many facultative anaerobes have only a soluble (NADlinked) malate dehydrogenase. Moraxella fwofi, like Micrococcus fysodeikticus (Cohn, 1958) and Acetobacter xylinium (Benziman & Galanter, 1964), has both a particulate and a soluble dehydrogenase (Jones, 1969; Jones & King, 1972). In this communication we report the purification and study of the NAD-dependent malate dehydrogenase of Moraxella Iwofi (N.C.I.B. 8250). Malate dehydrogenase (EC 1.1.1.37) was assayed by measuring the initial rate of decrease in EJIO resulting from the oxidation of NADH during the reduction of oxaloacetate or by the increase in EJIO resulting from the reverse reaction. Moraxella Iwofi was grown aerobically on a semi-synthetic medium with glutamate as carbon source to late exponential phase. Cells were disrupted by passage through a French pressure cell at 1.4 x lo9 Pa. The cell-free extract was separated into a particulate fraction and a supernatant fraction by centrifugation at 365000g at 4°C. The NAD+dependent malate dehydrogenase was present in the supernatant fraction, and this fraction was used for subsequent purification. The purification procedure involved ion-exchange chromatography with DEAEcellulose, preparative polyacrylamide-gel electrophoresis, followed by stepwise elution on a second DEAE-cellulose column. The preparation yielded a pure enzyme (single protein bands on 7.5,lO and 15 % polyacrylamide gels coincident with malate dehydrogenase activity, demonstrated by tetrazolium staining on replicate gels). The mol.wt. of the malate dehydrogenase was estimated as 61 OOO (by gel filtration on Sephadex G-100) or 60300 (by ultracentrifuge). Kinetic studies for the determination of the K,,, for oxaloacetate (3.87~ ~O-'M) and

Influences of growth substrates and oxygen on the electron transport system in Acinetobacter sp. HO1-N

“…Inhibition analysis. The effects of inhibitors on electron transport can provide data concerning the composition of the electron transport chain (13). Membranes prepared from Acinetobacter sp.…”