Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe
1993
An enzyme that catalyzes the asymmetric hydrolysis of Ap(4)A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose. Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap(4)A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap(4)A 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(4)A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 micromolar for Ap(4)A. Both forms of the enzyme asymmetrically hydrolyze Ap(4)A to AMP and ATP as determined by HPLC. Ap(4)A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 micromolar. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap(4)A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap(4)A-degradative enzyme. Fluoride, from 50 micromolar to 20 mM, has no significant effect on Ap(4)A hydrolase activity.
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