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THUNBERG [1920] observed that natural glutamic acid was oxidized in the presence of frog muscle. Weil-Malherbe [1936] was the first to prepare an enzyme extract which catalysed this oxidation but he did not resolve the system into its catalytic components. While this present analysis of the 1(+)glutamic dehydrogenase system was in progress Adler et al. [1937] described the properties of the same enzyme in liver. They foundthat coenzyme I was essential to the system and that the products of the reaction were a-ketoglutaric acid and ammonia. They also demonstrated the reversibility of the reaction. I. Preparation of the components The 1(+)glutamic dehydrogenase was prepared from liver or kidney of pig as follows. About 300g. liver or kidney are minced,in a Latapiemincer. To the mince are added 4 vol. cold acetone and the mixture is stirred for about 3 min., ifitered on a Buchner funnel and washed in turn with acetone and ether. The material on the filter paper is again mixed with 4 vol. acetone, filtered, washed with acetone and ether and spread out on paper to dry in a current of air. 10 g. of the dry powder are rubbed up with 100 ml. distilled water and the mixture centrifuged. The supernatant fluid after filtration is acidified with 10 % acetic acid to pH 4-6, the centrifuged precipitate washed with distilled water and finally suspended in 25 ml. M/10 phosphate buffer, pH 7*3. The enzyme keeps its activity in solution at 00 for 4-5 days. The acetone powder can be kept in vacuo for several weeks without loss of activity. Coenzyme I was prepared by the method outlined by Green et al. [1937]. II. The reaction with molecular oxygen A mixture of the dehydrogenase, coenzyme I and l(+)glutamic acid in the presence of a carrier such as pyocyanine takes up 02 readily. The rate of 02 uptake is dependent on the concentrations of the various reactants, i.e. dehydrogenase, coenzyme, substrate and carrier (cf. Figs. 1-4). As in other coenzyme systems the proportionality between the rate of oxidation and the concentrations of the different components holds only at low concentrations. Above some limiting concentration the system becomes saturated with the component in question. The Michaelis constant, i.e. the substrate concentration at which half the maximum velocity is reached is approximately M/133 (Fig. 3). Carriers which have been found to be active in the 1(+)glutamic acid system include pyocyanine, methylene blue, cytochromes a and b, flavinphosphate and flavoprotein from yeast (Table I). Cytochrome c in large concentrations (1.2 mg./ml.) has a small effect on the 02 uptake. The most efficient carrier (1378)

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