Prodan fluorescence reflects differences in nucleotide-induced conformational states in the myosin head and allows continuous visualization of the ATPase reactions.
The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.
Hiratsuka
T
T
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan. tashiaki@asahikawa-med.ac.jp
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D003570:Cytidine Triphosphate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007293:Inosine Triphosphate / Q000737:chemistry; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012265:Ribonucleotides / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014544:Uridine Triphosphate / Q000737:chemistry
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D012265:Ribonucleotides; D007293:Inosine Triphosphate; D003570:Cytidine Triphosphate; C038517:prodan; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D015081:2-Naphthylamine; D000251:Adenosine Triphosphatases; D014544:Uridine Triphosphate