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

Abstract

1. A nucleoside triphosphatase (EC 3.6.1.3) was solubilized from avian myeloblastosis virus after treatment with ethanol and sonication at an alkaline pH. The enzyme was purified by ammonium sulfate precipitation, chromatography on Bio-Gel A-0.5m, and sucrose density gradient centrifugation. 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 subunit structure of (α₂β₂γ δ ε (based on stain intensities), a molecular weight of about 314,000 was calculated. In sucrose density sedimentation, an s_20,w value of 19 S was observed, corresponding to a molecular weight of 650,000 suggesting formation of a dimer. 3. The enzyme required Ca²⁺ or Mg²⁺ 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, deoxycholate, 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, quantitatively precipitated and the antiserum-insensitive activity aws recovered in the precipitate.