Isolation and characterization of diadenosine tetraphosphate (Ap 4A) hydrolase from Schizosaccharomyces pombe

Abstract

An enzyme that catalyzes the asymmetric hydrolysis of Ap 4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH 4) 2SO 4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap 4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap 4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap 4A hydrolase activity. Both forms of the enzyme have a K m value in the range of 22 to 36 μM for Ap 4A. Both forms of the enzyme asymmetrically hydrolyze Ap 4A to AMP and ATP as determined by HPLC. Ap 4A is the optimal substate among several nucleotides and dinucleoside polyphosphates tested at 10 μM. A divalent metal cation is required for activity. Concentrations of P i below 30 mM stimulate Ap 4A hydrolase while higher concentrations inhibit the activity. P i is not a substrate for this Ap 4A-degradative enzyme. Fluoride, from 50 μM to 20 mM, has no significant effect on Ap 4A hydrolase activity.