119] l-glutamate dehydrogenases (yeast)

Abstract

Two types of glutamate dehydrogenases are found in yeast. One is TPN + and triphosphopyridine nucleotide (TPNH) specific, and the second utilizes only the dinitrophenyl (DPN) forms. Both enzymes may be purified from the same yeast extract, although the DPN enzyme is present in higher concentrations when the yeast is grown on glutamate. This chapter describes the preparations for both enzymes for the same extract from Fleischmann's yeast cakes. Both enzymes are assayed by following the oxidation of the reduced coenzyme. In crude extracts, there is endogenous oxidation of both diphosphopyridine nucleotide (DPNH) and TPNH. All the purification steps of the DPN and TPN glutamate dehydrogenases are generally carried out at 0–4°. Both enzymes may be purified from the same yeast extract. The extract is clarified by addition of 1/40 volume of 1 M MnCl 2 and allowed to stand for 10 minutes and then centrifuged at 9,000 rpm for 10 minutes. The clarified extract shows no change in protein or enzymatic activity, but this clarification facilitates further fractionation by ammonium sulfate.