The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Interaction of a New Fluorescent ATP Analogue with Skeletal Muscle Myosin Subfragment-1
Shinsaku MarutaYoshiaki MizukuraShigeru Chaen
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2002 Volume 131 Issue 6 Pages 905-911


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 was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using 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 fluorescence 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•AIF4- complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38×10-2 s-1 for BeFn and 1.1×10-3 s-1 for AlF4-. 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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