Purification and characterization of extracellular adenosine deaminase from a Streptomyces sp.

Abstract

Extracellular adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified from the culture filtrate of Streptomyces sp. J-845S by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-100, and CM-cellulose. The purified enzyme preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 90,000 by gel filtration with Sephadex G-100 and about 45,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme consisted of two subunits, and its isoelectric point was 7.8. The enzyme was stable at pH 3.5–5.5, the optimum pH for the reation was 5.0–6.0, and the optimum temperature was 55°C in the optimum pH range. Adenosine, 2′-deoxyadenosine, 2′,3′-isopropylidene adenosine, AMP, adenosine-3′-monophosphate, and cAMP were substrates of the enzyme, and the K m values for these compounds were 0.67, 0.22, 0.29, 0.17, 2.0, and 0.5 mM, respectively. The enzyme reaction was promoted by Fe 3+, and inhibited by Hg 2+, Ag +, and o-phenanthroline.