Multiple deoxyribonucleic acid dependent adenosine triphosphatases in FM3A cells. Characterization of an adenosine triphosphatase that prefers poly[d(A-T)] as cofactor

Abstract

Four chromatographically distinct DNA-dependent ATPases, B, C1, C2 and C3, were partially purified from mouse [mammary carcinoma] FM3A cell extracts. These ATPases are distinguished from each other by their physical and enzymological properties. DNA-dependent ATPases B, C1, C2 and C3 have sedimentation coefficients in 250 mM KCl of 5.5, 5.3, 7.3 and 3.4S, respectively. ATPases B, C2 and C3 hydrolyze dATP as efficiently as ATP, whereas C1 does not. ATPase B hydrolyzes other ribonucleoside triphosphates with relatively high efficiency as compared to the other 3 enzymes. ATPase C3 prefers poly[d(A-T)] to poly(dT) as cofactor, whereas the other 3 enzymes prefer poly(dT) to poly[d(A-T)]. Among the 4 ATPases, ATPase C3 was highly purified and characterized in detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction of ATPase C3 showed 2 major bands corresponding to MW of 66,000 and 63,000. The Km values of the enzyme for ATP and dATP are 0.53 and 0.86 mM, respectively. As cofactor, poly[d(A-T)] is the most effective among the DNA tested. Heat-denatured DNA and native DNA are also effective but used with less efficiency. Almost no or very little activity was detected with ribohomopolymers and oligonucleotides. The activity attained with poly(dT) and poly(dA) is 11 and 6% of that with heat-denatured DNA, respectively. When both polymers were added at a molar ratio 1 to 1, very high activity was obtained with these polymers. Little activity was observed by the combination of noncomplementary homopolymers such as poly(dT) and poly(dG).