Purification and Characterization of Diphosphopyridine Nucleosidase from Pig Brain

Abstract

Abstract Particulate diphosphopyridine nucleosidase from pig brain has been solubilized with trypsin and purified 8000-fold. The purified material appeared to be homogeneous in the ultracentrifuge; an s20,w value of 2.5 and a molecular weight of 26,000 were derived for the enzyme. The protein gave one precipitin band, indicative of one species of antigen, to a specific rabbit antibody. Less than 5% contaminant was present in the purified material as determined by cellulose acetate electrophoresis. Amino acid composition of the pig brain enzyme differed significantly from that of Bacillus subtilis diphosphopyridine nucleosidase. The purified enzyme and the acetone powder exhibited identical catalytic properties with respect to hydrolysis of diphosphopyridine nucleotide, 3-acetylpyridine exchange, and specificity for DPN+ and TPN+, as well as identical pH profiles. The trypsin-solubilized enzyme, and material solubilized with lipase, had molecular weights of 26,000 and 25,000 (±1,000), respectively, on Sephadex G-100. Both solubilized forms of the enzyme had the same electrophoretic mobility as determined by a staining technique developed to test for catalytic properties of mammalian diphosphopyridine nucleosidases. These results indicate that the purified material possesses the same catalytic properties present in the acetone powder and that solubilization with trypsin does not alter the native protein since it is identical with lipase-solubilized material.