Abstract

A new fluorescent ribose-modified ATP analogue, 2‘(3’)-O-{6-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoic-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1(S-1) was studied.NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP Fluorenscence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1-NBD-ADP-BeFn and S-1 NBD-ADP-A4 complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10−2 s−1for BeFn and 1.1 x 10−3 s−1 for AIF4. These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1-ADP-BeFn or AIF4 reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.

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