GREEN & BROSTEAUX [1936] and Green [1936, 2] showed that the lactic and malic dehydrogenases of animal tissues could react with oxygen only in presence of coenzyme I, a carrier and a ketone fixative. The dehydrogenase catalysed the transfer of hydrogen from the substrate to the coenzyme; in turn the reduced coenzyme reduced the carrier, and finally reduced carrier reacted with molecular oxygen. The function of the ketone fixative consisted in binding the keto-acid formed by the oxidation of either lactic or malic acid. The product of oxidation in both cases completely arrested the catalytic oxidation unless removed by the ketone reagent.Adrenaline, flavin and flavoprotein were the only substances occurring in animal tissues which were found capable of acting as carriers in the lactic and malic systems. The mechanism of the flavin and flavoprotein effects was perfectly clear. These substances were alternately reduced by the coenzyme and oxidized by molecular oxygen. This simple interpretation however failed to account for the adrenaline effect. The experiments of Green & Brosteaux [1936] and Green [1936, 2] showed clearly that the action of adrenaline as an oxidation carrier involved a complicated mechanism. The present communication deals with the analysis of the adrenaline effect in the lactic and malic systems of animal tissues. I. Experimental details The lactic and malic dehydrogenases were prepared from the heart muscle of pig by the method of Green & Brosteaux [1936]. The preparation of coenzyme I is also described in that paper.The manometric experiments were carried out in Barcroft differential manometers at 380. The reactions were started after equilibration by dislodging the Keilin cups containing adrenaline into the main portion of the manometer cups.The rate of shaking was 150 oscillations per min. The adrenaline solutions were prepared by dissolving the recrystallized base in water containing the theoretical amount of hydrochloric acid necessary for neutralization (final pH 5). These solutions were stable for weeks when kept at 00.II. The dependence of the adrenaline effect on the activity of the lactic and malic systems A mixture of enzyme, coenzyme I, malate (or lactate) and cyanide at pH 8 failed to take up any oxygen. With addition of adrenaline a vigorous uptake ensued. This effect was observed only when the complete malic or lactic system was present (cf . Tables I and II). If the enzyme, coenzyme, substrate or fixative was omitted, adrenaline had no influence on the oxygen uptake.
] have considered some of the properties of the glycerophosphate dehydrogenase. There has not been however any systematic investigation of this enzyme, and there are no data available as to the method of preparation, the conditions for maximum activity, the nature of the oxidation product or the mechanism of the reaction with molecular oxygen. I. Preparation of the enzyme. The dissected skeletal muscles of a freshly killed rabbit were passed twice through a coarse meat mincer, and washed exhaustively with tap water. The washed mince was mixed with sand and 500 ml. distilled water and ground to a paste in a mechanical mortar. The sand and insoluble debris were filtered off through muslin. 50 ml. of M/10 acetate buffer of PH 4-6 were added to the filtrate, and the precipitate was centrifuged. The supernatant fluid was discarded and the precipitate was resuspended in 100 ml. M/5 phosphate buffer of pl1 7-2. The enzyme suspension retains the bulk of its activity for a period of 10 days if kept at 00. There is a definite fall in activity even at this low temperature. The precipitate can also be dried, in vacuo. The enzyme in the dried
THE following contribution is the fifth of a series devoted to the detailed analysis of individual dehydrogenase systems [cf. Ogston & Green, 1935, 1, 2; Green, 1936; Green & Brosteaux, 1936]. The immediate goal of these studies is a knowledge of the components of the various dehydrogenase systems, and anunderstanding of the mechanism by which these systems react with molecular oxygen. The ultimate goal is the reconstruction in vitro of series of parallel and coupled oxidations of the type living cells perform. The means by which the living cell integrates and coordinates the various oxidation systems has yet to be discovered. All of the known dehydrogenases may be divided into three groups, viz. aerobic oxidases, cytochrome systems and coenzyme systems. Keilin & Hartree [1936] showed that the aerobic oxidases as a class react directly with molecular oxygen without requiring either a carrier or a coenzyme and produce H202 which can be utilized in coupled oxidations. The aerobic oxidases of animal tissues, e.g. uricase, amino-acid oxidase and xanthine oxidase are not generally distributed among the various organs and tissues but are restricted almost
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