Council for a whole time grant. We should like to express our gratitude to Prof. E. L. Hirst for specimens of d-araboascorbic and d-glucoascorbic acids, and to Prof. T. Reichstein for a specimen of 6-desoxy-l-ascorbic acid. Thanks are also due to Roche Products Ltd. for a gift of 1-ascorbic acid.
A strain of Aspergillus fumigatus Fresenius, isolated from spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown to be melilotic acid (I) [Formula: see text] coumaric acid (IV) [Formula: see text] beta-hydroxymelilotic acid (II) [Formula: see text] beta-oxomelilotic acid (III) [Formula: see text] 4-hydroxycoumarin (VI), on the basis of (1) studies on the formation of postulated intermediates, (2) experiments with isotopically labelled materials and (3) sequential enzyme induction. In the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric acid: there is no evidence, however, that this lies on the normal metabolic pathway.
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
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