Affinity Labeling of the Myosin ATPase with Ribose-Modified Fluorescent Nucleotides and Vanadate

Abstract

Ribose-modified fluorescent nucleotide analogs, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives of AT (D) P, dAT (D) P, CT (D) P, UT (D) P, IT (D) P, and GT (D) P, were synthesized for use as substrates and affinity labels for the myosin ATPase [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. None of the fluorescent nucleoside triphosphate (NTP) analogs was significantly different from the corresponding natural NTP in its ability to support superprecipitation of actomyosin. When fluorescent and natural NTPs were used as substrates for the myosin subfragment-1 (S-1) ATPase in the presence of 1mM vanadate ion (V₁), a slight initial inhibition of the S-1 NTPase was followed by progressive inhibition to more than 60% over a period of 1h. The apparent second-order rate constants were 0.14-0.44M^-1•s^-1, suggesting the formation of the inactive fluorescent NDP-labeled S-1. After incubation of S-1 with the nucleoside diphosphate (NDP) analog in the presence of V₁, the resultant fluorescent NDP-labeled S-1 was isolated free of unbound V₁ and the analog by gel filtration. The isolated complexes had stoichiometries of 0.6-1.1 NDP analog per S-1 active site. Native polyacrylamide gel electrophoresis revealed conveniently that the NDP analog is associated with S-1 as indicated by two intense fluorescent bands corresponding to S-1 isozymes. On dissociating gels, the analog was released from S-1, suggesting that the labeled S-1 is held together by strong secondary forces rather than covalent bonds. Neither nucleoside monophosphate analogs nor P₁ could substitute for NDP analogs or V₁, respectively, for the labeling. Labeling was abolished by excess ATP and ADP but not by AMP. The results indicate that these nucleotide analogs and V₁ function together as fluorescent affinity labels for the myosin active site. It is further concluded that the pyrophosphate bond of NDP is essential for the formation of the stable myosin-NDP-V₁ complex but its 2'- and 3'-hydroxyl groups are not.