Solubilization, purification, and characterization of a nucleoside triphosphatase from avian myeloblastosis virus.

Abstract

1. A nucleoside triphosphatase (EC 3.6.1.3) was solubi- lized from avian myeloblastosis virus after treatment with ethanol and sonication at an alkaline pH. The enzyme was purified by ammonium sulfate precipitation, chromatogra- phy on Bio-Gel A-0.5m, and sucrose density gradient cen- trifugation. 2. In sodium dodecyl sulfate-acrylamide gels, the protein dissociated into five subunits with molecular weights of 62,000, 60,000, 28,000, 24,000, and 18,000. Assuming a sub- unit structure of (~.&y 6 E (based on stain intensities), a molecular weight of about 314,000 was calculated. In su- crose density sedimentation, an s~,,,~,. value of 19 S was observed, corresponding to a molecular weight of 650,000 suggesting formation of a dimer. 3. The enzyme required Ca’+ or MgZ+ for activity and hydrolyzed ATP, GTP, CTP, UTP, and ITP at a similar rate. ADP was hydrolyzed at one-tenth the rate of ATP and no significant hydrolysis of AMP or PP, was detected. 4. The enzyme activity was resistant to a variety of inhibitors of mitochondrial and (Na+-K+)-ATPase but was sensitive to mercurials and N, N ‘-dicyclohexylcarbodiimide (DCCD). Treatment of the enzyme with phospholipase A stimulated ATPase activity and reduced its sensitivity to DCCD. The enzyme was extremely sensitive to most of the ionic and nonionic detergents such as cholate, deoxycho- late, Triton X-100, Tween 80, NP-40, Lubrol, or lysolecithin, which explains the failure of previous attempts to solubilize the enzyme. Trypsin inactivated the ATPase activity which was protected in the presence of ATP or EDTA. 5. A rabbit antiserum against the enzyme inhibited the activity by 40 to 50%. The protein was, however, quantita- tively precipitated and the antiserum-insensitive activity was recovered in the precipitate.