KINETIC AND SPECTROSCOPIC CHARACTERIZATION OF FLUORESCENT RIBOSE-MODIFIED ATP ANALOGS UPON INTERACTION WITH SKELETAL-MUSCLE MYOSIN SUBFRAGMENT-1

Citation
Pb. Conibear et al., KINETIC AND SPECTROSCOPIC CHARACTERIZATION OF FLUORESCENT RIBOSE-MODIFIED ATP ANALOGS UPON INTERACTION WITH SKELETAL-MUSCLE MYOSIN SUBFRAGMENT-1, Biochemistry, 35(7), 1996, pp. 2299-2308
Citations number
50
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
7
Year of publication
1996
Pages
2299 - 2308
Database
ISI
SICI code
0006-2960(1996)35:7<2299:KASCOF>2.0.ZU;2-7
Abstract
The interaction of the fluorescent ATP analog 2-[3-(5-fluoresceinyl)th ioureido]-ethyl]carbamoyl] adenosine 5'-triphosphate (FEDA-ATP) with r abbit skeletal myosin subfragment 1 (S1) and acto-S1 was studied. This and related ATP analogs are potentially useful for determination of t he ATPase activity of single myosin filaments using fluorescence micro scopy [Sowerby et al. (1993) J. Mel. Biol. 234, 114-123]. However, it is neccesary that such analogs mimic ATP in their kinetics of turnover . The apparent second-order association rate constants for FEDA-ATP bi nding to S1 and for FEDA-ATP-induced dissociation of acto-S1 are about 4 times slower than those for ATP. As with ATP, the hydrolysis step i s fast, so that the M . FEDA-ADP . P-i complex is the major steady-sta te intermediate. The turnover rate is the same for the 2' and 3' FEDA- ATP derivatives and similar to that of ATP itself. The dissociation ra te constant for FEDA-ADP from S1 is identical to that for ADP. Actin-a ctivated turnover is comparable for both FEDA-ATP and ATP. The corresp onding rhodamine and sulfoindocyanine, Cy3.18 (Cy3), derivatives also appear to be reasonable analogs. FEDA-ATP binding leads to a 25-40% re duction in fluorescein fluorescence. Spectral properties of the bound nucleotide were explored by trapping FEDA-ADP as its aluminum fluoride complex. The fluorescence quenching is a consequence of a reduction i n both absorbance and excited-state lifetime, but there is little chan ge in spectral shape.