Two Nucleic Acid-dependent Nucleoside Triphosphate Phosphohydrolases from Vaccinia Virus

Two nucleic acid-dependent nucleoside triphosphate phosphohydrolases have been found in vaccinia virus cores. Both enzymes hydrolyze ATP to ADP and Pi in the presence of nucleic acid. The enzymes were solubilized from purified viral cores by treatment with sulfhydryl-reducing agent, salt, and sodium deoxycholate in a Tris buffer at alkaline pH. DNA-free viral extracts were prepared by adsorption of viral DNA to DEAE-cellulose columns in the presence of high salt, and the two enzymes were purified by DNA-cellulose chromatography. Phosphohydrolase I was purified 300- to 400-fold with yields of 80 to 90 % and is approximately 95 % pure. A molecular weight of approximately 61,000 was calculated for the native enzyme using Sephadex gel chromatography and sucrose gradient sedimentation. A molecular weight value of 68,000 was obtained under denaturing conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that phosphohydrolase I is a monomeric enzyme. The smaller amount of phosphohydrolase II prevented accurate determinations of specific activity or purity. The molecular weight of the native enzyme was calculated to be 68,000 and it may also consist of a single polypeptide. Nucleic acids were required by phosphohydrolase I and II for the hydrolysis of ATP, and optimal activities were obtained at neutral pH in the presence of divalent cations. The hydrolysis of ATP resulted in the production of stoichiometric amounts of ADP and Pi. The Km values of phosphohydrolases I and II were 1.4 x 10-4 m and 6.4 x 10-4 m, respectively.