Preparation of a new fluorescent analog of ATP, 2'-(5-dimethylaminonaphthalene-1-sulfonyl)amino-2'-deoxy ATP, and its interactions with myosin and actomyosin.
A fluorescent ATP analog, 2'-(5-dimethylaminonaphthalene-1-sulfonyl)amino-2'-deoxy ATP (DNS-ATP), was synthesized. In water, the wavelengths of maximum excitation were 260 and 340 nm, and that of maximum emission was 554 nm. The fluorescence quantum yield with excitation at 340 nm was 0.052. In 80% dioxane-20% water solution, the wavelength of the maximum emission shifted to 527 nm and the quantum yield was about 5.4 times in water. When DNS-ATP was mixed with HMM in the presence of Mg2+ ions, the fluorescence intensity of DNS-ATP was enhanced by about 30%, and the wavelength of maximum emission shifted to 545 nm. The observed second-order rate constant for the change in fluorescence intensity after adding DNS-ATP to HMM was 1.6 x 10(-7) M-1 . s-1, while the observed first-order rate constant for its recovery was 0.17 s-1. When the HMM DNS-ATPase reaction was measured in terms of the TCA-Pi liberation, 1 mol of initial burst of Pi liberation per mol of myosin was observed. In 50 mM KCl and at 20 degrees C, the rate of the HMM DNS-ATPase reaction was increased by F-actin from 0.4 to 1.15 s-1 (in 3 mg/ml F-actin). The observed dissociation constant for the binding of DNS-ATP with HMM increased from 1.2 to 20 microM in the presence of 5 mg/ml F-actin. However, the extent of change in fluorescence intensity at infinite concentration of DNS-ATP was unaffected by the presence of F-actin.
D000205:Actomyosin / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006868:Hydrolysis; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; C032569:dansylamino deoxy-ATP; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins