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Distinct Structures of ATP and GTP Complexes in the Myosin Atpase a Variety of Ntps Other Than ATP (6-16). How Ever, GTP And

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Distinct Structures of ATP and GTP Complexes in the Myosin Atpase a Variety of Ntps Other Than ATP (6-16). How Ever, GTP And J. Biochem. 96, 155-162 (1984) Distinct Structures of ATP and GTP Complexes in the Myosin ATPase Toshiaki HIRATSUKA Department of Chemistry, Asahikawa Medical College Asahikawa, Hokkaido 078-11 Received for publication, February 10, 1984 The active site of the myosin subfragment-1 ATPase was affinity-labeled with ribose modified fluorescent analogs of ADP, dADP, CDP, UDP, IDP, and GDP in com bination with vanadate, forming a stable myosin-nucleoside diphosphate-vanadate complex that is analogous to the normal myosin-ADP-Pi intermediate [Hiratsuka, T. (1984) J. Biochem. 96, 147-154]. Labeled enzyme was isolated free of unbound analog and vanadate, and fluorescent properties of the fluorophore at the active site were examined. Fluorescence emission and acrylamide quenching studies re vealed that the hydrophobicity of environment around the fluorophore and the degree of its burial in the protein vary with the base structure of NDP. It was found that the fluorophore of ADP analog is most buried into the protein, while that of the GDP analog is least buried. The results suggest that the deep burial of ATP into the myosin active site is essential for muscle contraction. Energy transducing involving nucleoside triphos a variety of NTPs other than ATP (6-16). How phate (NTP) hydrolysis is usually linked to ATP ever, GTP and other NTPs cannot substitute for hydrolysis. However, this is not always the case. ATP completely in its ability to support super Energy from GTP is also used to control the con precipitation of actomyosin (12-14), to contract formation of proteins in a large number of bio and relax myofibrils (6, 14), and to protect the chemical processes, including protein synthesis (1), active site of myosin against thermal denaturation assembly of tubulin (2, 3), activation and inhibi (7). Furthermore, a mechanism of NTPase differ tion of adenylate cyclase (4), and rhodopsin-cata ent from that of ATPase has been proposed using lyzed cGMP phosphodiesterase (5). Therefore, it GTP (15) and ITP (16) as substrates for myosir is of interest to understand what are the special subfragment-l (S-1) and heavy meromyosin, re structural properties of ATP compared with GTP spectively. These results suggest that the myosir in biological systems. active site recognizes the base structure of the NTP The myosin ATPase also hydrolyzes GTP and molecule. However, it is unknown how NTP is buried into the crevice of the active site. One approach to this problem is the use of a Abbreviations: S-1, myosin subfragment-1; V;, ortho. vanadate; NDP, nucleoside diphosphate; NTP, nucleo reporter-labeled nucleotide analog which mimics side triphosphate; Ant, 3•Œ-O-anthraniloyl; Mant the natural nucleotide (8-10). We have previously 3•Œ-O-(N-methylanthraniloyl). synthesized various ribose-modified fluorescent nu- Vol. 96, No. 1, 1984 155 156 T . HIRATSUKA cleotide analogs, 3•Œ-O-anthraniloyl and 3•Œ-O-(N (2-3 ml) in buffer A over a Sephadex G-25 column methylanthraniloyl) derivatives (11, 13). For these (1.8•~19 cm). The amount of NDP analog la analogs, a fluorophore which is smaller than most beled to S-1 was estimated from the difference others is attached to the 3•Œ-hydroxyl group of the absorption spectra (13). ribose of nucleotide, leaving the base and phos All fluorescence measurements were carried phoryl moieties intact, and producing only minor out in buffer A at 25•Ž in a Hitachi fluorescence perturbation in enzyme reactions (11, 13, 17). In spectrophotometer, Model MPF-4, equipped with addition, their fluorescence emission is sensitive to a corrected spectra accessory (11, 17). The ab environmental changes, making these analogs ex solute quantum yields of fluorescent derivatives tremely good fluorescent probes for proteins (18 , were measured as described previously (11, 17, 19). 26), using quinine sulfate in 0.1 N H2SO4 as a In the preceding paper (13) , we showed that standard of 0.70 (27). The quenching of Ant and these NTP analogs with various base structures Mant fluorescence by acrylamide was measured by are useful as fluorescent substrates for the myosin adding the quencher to a solution of labeled S-1 ATPase, since they have biological activities similar (0.1 mg/ml). The collisional quenching constant to those of the corresponding natural NTP. Fur for fluorescence (KQ) was calculated from Stern thermore, the analogs of nucleoside diphosphate Volmer equation: (NDP) and vanadate ion (Vi) function together as affinity labels for the S-1 active site as well as ADP and Vi (20), forming stable S-1-fluorescent NDP-Vi complexes. Since the myosin-ADP-Vi complex is now believed to be a stable analog of where ƒÑ0 and ƒÑ are the fluorescence lifetimes in the the myosin-ADP-P1 intermediate (21, 22), the iso absence and presence of quencher (Q), F0 and F lated S-1-fluorescent NDP-Vi complexes should represent the fluorescence intensities at the emis be useful to investigate the base-specific structures sion maximum in the absence and presence of Q, of myosin-nucleotide complexes. In the present and kq is the rate constant for the quenching paper, the fluorescent properties of S-1 labeled reaction. Evidence for a collisional rather than with ADP analogs are compared with those of S-1 static quenching process by acrylamide was ob labeled with analogs of dADP, CDP, UDP, IDP, tained by measurement of ƒÑo and ƒÑ with free and and GDP. actin-bound Ant-ATP and Mant-ATP (Mihashi, K., unpublished results). Thus, the apparent value MATERIALS AND METHODS of KQ is inversely proportional to the degree of burial of the fluorophore (28). Excitation and Materials-Rabbit skeletal myosin was pre emission wavelengths were 330 nm and 428 nm , pared by the method of Perry with slight modi respectively, for Ant analogs, and 350 nm and 446 fication (23). S-1 was prepared by chymotryptic nm for Mant ones. The slit widths on the excita digestion of myosin (24) with modification as de tion and emission monochromators were 5 run. scribed by Weeds and Taylor (25), and used within All measurements were completed within 5 h after 10 days. V2O5 was purchased from Nakarai gel filtration of samples. Chemical Co. Stock solutions of Vi were prepared The concentration of S-1 was determined from as described by Goodno (20). Ant and Mant the extinctioncoefficient (A1%1cm) at 280 nm of 7.5 analogs of NDP were prepared as described pre (29). Protein concentrationsof labeled S-1 were viously (11, 13). Other reagents were of reagent determinedby the biuret method(30) , standardized or biochemical research grade. usingA1%1cm of unlabeled S-1. Methods-Affinity labeling of S-1 was carried Concentrations of Ant and Mant analogs were out at 25•Ž in buffer A (90 mM NaCl, 5 mM determined by absorption at 332 nm (ƒÃ=4,700 MgCl2, 20 mM Tris-HCl, pH 8.5) as described pre M-1•Ecm-1) and at 356 nm (ƒÃ=5,800M-1•Ecm-1) , viously (13). Before fluorescence measurements respectively (11, 13). and ATPase assays, free Vi and fluorescent NDP were removed by passage of the reaction mixture J. Biochem. STRUCTURES OF THE MYOSIN-ATP AND -GTP COMPLEXES 157 beled to S-1-The effect of S-1 on the fluorescent RESULTS properties of Ant-ADP was also examined in 90 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl (pH 8.5) Fluorescent Properties of NDP Analogs-Ab at 25•Ž. The fluorescence spectrum of Ant-ADP sorption spectra of Ant and Mant analogs of NDP in the presence of S-1 (0.4 mg/ml) was recorded exhibit a broad band of the fluorophore centered and compared with the spectrum of the free ana at 332 nm and 356 nm, respectively, regardless of log. No spectral shift occurred in the emission their base structures (11, 13, 17). Upon excita maximum of the analog (428 nm) upon addition tion at 330-350 nm both Ant and Mant analogs of S-1. However, a large fluorescence enhance exhibit maximum fluorescence emission at 430 ment was observed in the presence of S-1. Fluo 445 nm in aqueous solution and at 410-430 nm in rescence titration data (not shown) indicated a organic solvents (11, 17). The potential usefulness 1.7-fold enhancement at saturation level (12 ƒÊM of these analogs as fluorescent probes of hydro Ant-ADP). phobic microenvironments is illustrated in Fig. 1. Although almost no spectral shift was observed in TABLE I. Quantum yields and stoichiometries of emission maxima (428 nm for Ant-ADP and 446 fluorescent NDP labeled to S-1. After incubation of nm for Mant-NDPs), quantum yields of Ant-ADP S-1 (2 mg/ml) with fluorescent NDP (1 mM) and Vi and Mant-NDPs increase approximately 2 and (1 mM) in buffer A (5 mM MgCl2, 90 mM NaCl, 20 mM 1.7-fold, respectively, in going from water to 20% Tris-HCl, pH 8.5) for 1 h at 25•Ž, the labeled-S-1 was isolated free of unbound Vi and NDP by gel filtration ethanol. Ant-ADP demonstrates more sensitivity on a Sephadex G-25 column (1.8•~19 cm) (13). Stoi toward the polarity of the environment than Mant chiometry of the fluorescent NDP labeled to S-1 was NDPs. Plots of QE/Qw vs. ethanol % were found determined from the difference absorption spectra be to be linear up to 18% concentration of ethanol tween labeled and unlabeled S-1. in both cases; QE and Qw were quantum yields of analog in ethanol and water, respectively. There was no significant difference in fluorescent prop erties between Mant-NDPs regardless of their base structures (see Table I).
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