Zur Kenntnis der Co-Enzymspezifität von Dehydrasen. I. Malico- und Glucose-dehydrase

Abstract

The anerodehydrogenases were characterized by a very weakly developed coenzyme specificity. Malic apodehydrogenase and glucose dehydrogenase were prepared by saturating the dialyzed H2O-acetone extract of horse liver with (NH4)2SO4 twice reprecipitating the precipitate obtained with half saturated (NH4)2SO4 and dialyzing the final product against 1% (NH4)2SO4 for 2 days in the ice box. A clear yellow enzyme soln. was obtained. Malic dehydrogenase was prepared from germinating peas by grinding and extracting with 21/4 times their wt. of M/10 Na2HPOi soln. The yellow-green soln. was dialyzed overnight against tap H2O. The enzyme was also prepared by dialyzing the maceration juice of bakers'' yeast and by repeatedly freezing Escherichia coli in liquid air, extracting the mixture with phosphate soln., pH 7.5, and dialyzing the deep yellow soln. 36 hrs. in the ice box. Glucose dehydrogenase was also prepared from the liver of cattle, sheep, and goats. Codehydrogenase I (cozymase or di-phosphopyridine nucleotide) was obtained from yeast and codehydrogenase II (triphosphopyridine nucleotide) by phosphorylating cozymase. The reaction mixtures usually contained 0.5 ml. M/2 d,l-Na2 malate, 0.5 ml. M/5 phosphate buffer, pH 7.5, 0.5 ml. M/5 semicarbazide, 0.15-1 ml. enzyme soln., 025 ml. M/2000 methylene blue soln., and 6-250 meg. coenzyme. The glucose reaction mixtures did not contain semicarbazide. The expts. were carried out at 30[degree] according to the Thunberg method. The malic dehydrogenase from liver, yeast and E. coli was activated specifically by codehydrogenase I, while the enzyme from peas was active in the presence of both codehydrogenase I and II. The glucose apodehydrogenase from liver did not show coenzyme double specificity, but was activated only by codehydrogenase I.