The purification of malic dehydrogenase from shrimp muscle



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The procedures for purifying the malic dehydrogenase isoenzymes from brown shrimp muscle have been investigated, in order, eventually, to study differences in cellular distributions, kinetic properties, molecular weights, and association-dissociation behavior. A study of these differences is important in comparing species of edible shrimp in order to determine the distribution in postlarvae samples, in comparing vertebrates and invertebrates in evolutionary studies, and in studying molecular control mechanisms. The purification of MDH isoenzymes by ammonium sulfate fractionation was successful in that most of the enzyme activity was concentrated in the 50-60% fraction and in that enzyme assays of polyacrylamide gels showed different molecular forms of MDH present in different fractions. The purification by ion-exchange chromatography was not as successful, but the groundwork was laid for further purification by this method. DEAE columns at pH 7.5 (phosphate buffer) remove other proteins from the ammonium sulfate fraction, 50-60%, so that although the molecular forms of MDH are not separated by the column, the resolution of the bands on the polyacrylamide gels is much sharper. Thus, the DEAE cellulose is useful in the purification of the MDH isoenzymes from the other enzymes and proteins present.