Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1.
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.BeF(n) and S-1.NBD-ADP.AlF(4)(-) 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(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.
Maruta
Shinsaku
S
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan. shinsaku@t.soka.ac.jp
Mizukura
Yoshiaki
Y
Chaen
Shigeru
S
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate