A diphosphopyridine nucleotidase and its protein inhibitor from Mycobacterium butyricum.

The diphosphopyridine nucleotidase exchange reaction, whereby isonicotinic acid hydrazide can replace nicotinamide in the DPN’ molecule, was demonstrated by Zatman el al. (1). Since this reaction provided a possible explanation for the growth inhibition of Mycobacteria by isonicotinic acid hydrazide, methods for measuring DPN in Mywbacteria were investigated. It was found that acetone powder extracts treated with 5 per cent trichloroacetic acid yield DPN, although after the extracts in 0.1 M KC1 were boiled to remove the protein, no DPN could be demonstrated to be present. Swartz, Kaplan, and Frech in this laboratory observed that boiled extracts of Proteus vulgaris would hydrolyze DPN at the pyrophosphate linkage but unboiled extracts were devoid of activity. They found further that the P. vu&zti.s system contained a heat-stable enzyme and a heat-labile inhibitor (2). Examination of an extract of Mycobacterium butytitim revealed that it contained a “heat-activated” enzyme which cleaves DPN at the nicotinamide-ribose linkage as well as a heat-labile inhibitor of this reaction. These extracts also differed from the P. vulgaris extracts in having a much greater excess of free inhibitor over that combined with the enzyme. A preliminary report on the M. butyricum DPNase and its inhibitor has been presented (3). The purpose of this paper is to describe the characteristics of, and the reaction between, the partially purified DPNase and the inhibitor.