Diacetilo reductasa de tejidos animales; purificación y estudios cinéticos de la procedente de hígado bovino

Abstract

lt is shown for first time that diacety 1 reductase is pressent in a variety of animal tissues. Its intracellular distribution is studied, showing its concentration in the soluble fraction, although there is sorne activity in mitochondria and other fractions.
Diactetyl reductase from beef liver has been partially purificd by a procedure involving acetone precipitation of acetone cakes water extracts and chromatography on DEAE-22-cellulose. By this method a 4 % yield of total activity is obtained with a 250 fold increase in specific activity over beef liver water extracts.
Enzyme preparations purified by acetone fractionation are highly specific for one of the substrates, diacetyl (butane-2-3-dione ), which can not be replaced by pentane-3-one, pentane-2-4-dione, hexane-2-5-dione, pyruvate, oxaloacetate. (J. -ketoglutarate or acetoin (acetyl-methyl-carbinol), but not for the other: NADPH can ben substituted for NADH. Enzyme activity shows a maximum at pH 6.1 and does not require metallic ions. At pH 6.1 the reaction in the diacetyl-acetoin direction follows Arrhenius law at least from 3.5°C. to 28-30°C.; activation energy has been estimated to be 14,400 cal./mol. It has not been possible to detect any activity in the backward direction (acetoin-diacetyl).
Sephadex gel filtration resolved the enzyme preparations in two peaks of activity; the molecular weights of the enzymatic species were estirnated as 26.000 and 76.000. The ratio of the peaks size varied with the eluant (phosphate buffer pH 6.3) molarity and this is taken as indicative that diacetyl reductase can be in two different states of association (as a monomer and a trimer ), the equilibrium being somehow regulated by the ionic strength of the buffer.
An extensive kinetic study was performed in order to elucidate the reaction mechanism. lt has been concluded that it follows an ordered mechanism but it has not been possible to distinguish between classical and Theorell-Chance variants.