Fractionation by deae-cellulose column chromatography and some properties of phosphodiesterase extracted from an andosol under forest

Abstract

Abstract An extract prepared from Andosol under forest was treated with protamine sulfate resulting in the precipitation and removal of brown-colored compounds in the extract. The protamine-treated extract was applied to a DEAE-cellulose chromatography column. Seven fractions with phosphodiesterase activity were obtained by stepwise elution with KCl (0-0.8 M) solution prepared in 0.02 M Tris-HCl buffer (pH 7.4). The fraction eluted by 0.2 M KCl was the largest for phosphodiesterase activity. Each fraction had a hydrolytic activity toward bis( p-nitrophenyl)phosphate, deoxythymidine-3′-p-nitrophenyl phosphate, deoxythymidine-5′-p-nitrophenyl phosphate and p-nitrophenyl phosphate. The artificial substrate with the nucleotide moiety was a better substrate than that without nucleotide moiety. Each fraction showed a hydrolytic activity for DNA and RNA. Optimum pH for the phosphodiesterase activity of each fraction ranged from 5.0-6.0. Michaelis constant of each fraction for bis-(p-nitrophenyl)phosphate ranged from 7.7 × 10-4 to 1.9 × 10-3 M. These properties of the fractions were compared with those of known enzymes which were reported to hydrolyze bis-(p-nitrophenyl)phosphate.