A Diphosphopyridine Nucleotide Oxidase in Mung Bean Seedlings

Abstract

Although the electrolytically obtained DPNH was not completely oxidized by usual dehydrogenases or diaphorases, one of the authors noticed that its absqrption band at 340 m,u disappeared completely when it was incubated with the extract of mung been seedlings. The reaction was found to be stimulated by the addition of methylene blue, and the product was identified as DPN. Thus, the reaction resembled that of diaphorase, although it was less specific to the configuration of DPNH. But unlike usual diaphorase, it required a cofactor, which was neither flavins nor metallic ion, but an unidentified acidic substance. General properties of the enzyme and the cofactor are reported in this article.

A dimer of diphosphopyridine nucleotide

“…The dimer was oxidized to DPN+ by the mung bean enzyme in the presence of a heat-stable factor in the bean seedlings. Details need not be given because the results are essentially the same as those obtained by Kono and Suekane (1958). The reader should note, however, that Kono incorrectly considered the dimer to be a dihydropyridine isomeric with DPNH.…”

“…The enzyme catalyzing the dimer oxidation was partially purified by differential precipitation with ammonium sulfate and by electrophoresis; the oxidant (Kono's cofactor) was not identified (Kono and Suekane, 1958).…”

“…Likewise, Kono reported partial reduction to DPNH (Kono, 1957; Kono and Nakamura, 1958); again, the coenzyrnatically inactive portion of the reduction mixture resembled the dimer reported here. In a following paper (Kono and Suekane, 1958), it was stated that the reduction led to a mixture of the 1,2-, 1,4-, and 1,6-dihydropyridines. Interestingly, an enzyme system was found in etiolated mung bean seedlings which reoxidized the dimer to DPN+, although of course it was incorrectly credited with the oxidation of the isomeric dihydropyridines (Kono and Suekane, 1958).…”

Electrochemical Reduction of Diphosphopyridine Nucleotide*

“…The significance of this unfortunately escaped Kono because he had no way of knowing that the "dimer" was present in his electrolytic DPNH preparations. The enzyme was not found in extracts of spinach, turnip, yeast, pig liver, or pig heart muscle (Kono and Suekane, 1958).…”

“…Kono (1957) noted that a fraction obtained from etiolated mung bean seedlings, unlike yeast alcohol, lactic acid, or malic acid dehydrogenases, was capable of eliminating completely the 340 µ absorption band of the electrolyzed solution. In a later paper (Kono and Suekane, 1958), an ammonium sulfate protein fraction obtained from an extract of the homogenized seedlings was studied further, and an electrophoretic fraction of much higher specific activity was obtained. Kono and Suekane state: "The enzyme differs from the other enzyme (diaphorase) in two characters.…”