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• W2061340145 abstract "To obtain a consistent view of the nucleotide-induced conformational changes around Cys697 (SH2) and Cys707 (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotide-induced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment. To obtain a consistent view of the nucleotide-induced conformational changes around Cys697 (SH2) and Cys707 (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotide-induced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment. myosin subfragment-1 6-acryloyl-2-dimethylaminonaphthalene 6-bromoacetyl-2-dimethylaminonaphthalene 6-propionyl-2-dimethylaminonaphthalene N-(iodoacetyl)-N′-(5-sulfo-1-naphthyl)ethylenediamine 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid adenyl-5′-yl imidodiphosphate polyacrylamide gel electrophoresis iodoacetamide The structural element of the molecular motor myosin is the subfragment-1 (S-1)1 moiety that contains the sites responsible for the ATP hydrolysis and binding of actin (1Mueller H. Perry S.V. Biochem. J. 1962; 85: 431-439Crossref PubMed Scopus (96) Google Scholar). A striking feature of the S-1 structure is a long helix spanning 85 Å that is stabilized by interactions with the myosin light chains (2Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1877) Google Scholar). It has been proposed that this light chain-binding domain acts as a semi rigid “lever arm” to amplify and transmit conformational changes in the ATP and actin binding sites of S-1 (3Lowey S. Waller G.S. Trybus K.M. Nature. 1993; 365: 454-456Crossref PubMed Scopus (268) Google Scholar, 4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar, 5Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. Nature. 1995; 378: 748-751Crossref PubMed Scopus (341) Google Scholar, 6Rayment I. J. Biol. Chem. 1996; 271: 15850-15853Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Uyeda T.Q.P. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar, 8Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 9Goldman Y.E. Cell. 1998; 93: 1-4Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Cys707 (SH1) and Cys697 (SH2) are located on the two different helixes in the C-terminal segment of the S-1 catalytic domain and separated from one another by 19 Å (2Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1877) Google Scholar). The two helixes are kinked at a conserved glycine residue (Gly699) found in a bend joining two helixes. Recent experiments have led to a conclusion that the fulcrum point for the swinging motion of the lever arm is in the vicinity of the SH1-SH2 region (4Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar, 5Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. Nature. 1995; 378: 748-751Crossref PubMed Scopus (341) Google Scholar, 6Rayment I. J. Biol. Chem. 1996; 271: 15850-15853Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Uyeda T.Q.P. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar, 8Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 9Goldman Y.E. Cell. 1998; 93: 1-4Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Furthermore, Gly699 (close to SH2) and Gly710 (close to SH1) have been proposed to act as pivot points or flexible hinges for such motion (8Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 10Patterson B. Ruppel K.M. Wu Y. Spudich J.A. J. Biol. Chem. 1997; 272: 27612-27617Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Kinose F. Wang S.X. Kidambi U.S. Moncman C.L. Winkelmann D.A. J. Cell Biol. 1996; 134: 895-909Crossref PubMed Scopus (85) Google Scholar). These data show the importance of motion in the SH1-SH2 region in the force generation cycle of myosin. However, the precise conformational changes of this region that are involved in lever arm movement are still unknown. One approach for understanding the involvement of the SH1-SH2 region in lever arm movement is a precise comparison of the individual conformational changes around SH1 and SH2 during ATPase cycle of S-1. Such a study could provide new insights into the mechanism of the lever arm movement in the generation of force. Although the conformational changes around SH1 have extensively been studied, little is known about those around SH2 (12Phan B.C. Peyser Y.M. Reisler E. Muhlrad A. Eur. J. Biochem. 1997; 243: 636-642Crossref PubMed Scopus (40) Google Scholar, 13Ajtai K. Burghardt T.P. Biochemistry. 1989; 28: 2204-2210Crossref PubMed Scopus (34) Google Scholar, 14Hiratsuka T. J. Biol. Chem. 1993; 268: 24742-24750Abstract Full Text PDF PubMed Google Scholar, 15Hiratsuka T. Biochemistry. 1988; 27: 4110-4114Crossref PubMed Scopus (10) Google Scholar). A recurrent question is whether SH2 is mobile or stationary. Several reports have suggested that SH2 is mobile (16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar, 17Rajasekharan K.N. Mayadevi M. Burke M. J. Biol. Chem. 1989; 264: 10810-10819Abstract Full Text PDF PubMed Google Scholar, 18Kasprazk A.A. Chaussepied P. Morales M.F. Biochemistry. 1989; 28: 9230-9238Crossref PubMed Scopus (19) Google Scholar). On the other hand, Xing and Cheung (19Xing J. Cheung H.C. Biochemistry. 1995; 34: 6475-6487Crossref PubMed Scopus (22) Google Scholar) have indicated that SH2 was relatively immobilized even in the presence of nucleotides. One approach to examine the mobility of SH2 is to attach a sensitive fluorescent probe covalently to SH2. However, little is known about a simple method for the fluorescent labeling of SH2 (20Reisler E. Methods Enzymol. 1982; 85: 84-93Crossref PubMed Scopus (82) Google Scholar). Thus, I have initiated a study of covalent fluorescent probes that react specifically with SH2. Several fluorescent probes were examined in an attempt to label only SH2. Of the compounds tested, 6-acryloyl-2-dimethylaminonaphthalene (AD) was chosen as a specific fluorescent label for SH2 (SchemeFS1). Furthermore, an attempt at the labeling of SH1 with the same fluorophore as that labeled with SH2 was made to obtain a consistent view of conformational changes around the two thiols. Finally 6-bromoacetyl-2-dimethylaminonaphthalene (BD) was chosen as a fluorescent label for SH1. The resultant AD and BD derivatives of S-1 have the same fluorophore, the 6-acyl-2-dimethylaminonaphthalene group, at either SH1 or SH2. Unlike the vast majority of the more commonly used fluorophores, the noncovalent fluorescent probe prodan, the parent fluorophore of AD and BD (Scheme FS1), should be particularly well suited to probe the surface of proteins by virtue of the exquisite sensitivity of the fluorophore to minor changes in the environment (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar, 22Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar, 23Marriott G. Zechel K. Jovin T.M. Biochemistry. 1988; 27: 6214-6220Crossref PubMed Scopus (45) Google Scholar, 24Hiratsuka T. Biochemistry. 1998; 37: 7167-7176Crossref PubMed Scopus (15) Google Scholar). It gives a 130-nm shift in the emission maximum from 401 nm in cyclohexane to 531 nm in water (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar). Thus the emission maximum of the fluorophore would truly be a polarity sensor for proteins. Furthermore, the half-widths of the emission spectra of the prodan fluorophore provide an indication of the accessibility of solvent molecule to the fluorophore attached to proteins (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar, 22Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar, 23Marriott G. Zechel K. Jovin T.M. Biochemistry. 1988; 27: 6214-6220Crossref PubMed Scopus (45) Google Scholar). Thus these remarkable properties of the prodan fluorophore are utilized in the present study to describe precise differences between SH2 and SH1 of S-1 in their environmental changes during ATPase cycle of S-1. AD and BD were purchased from Molecular Probes. IAEDANS was from Aldrich. IAA was from Wako Pure Chemicals. α-Chymotrypsin, diphenylcarbamyl chloride-treated trypsin, soybean trypsin inhibitor, and AMP-PNP were from Sigma. ATP and ADP were from Kohjin. Other reagents were reagent or biochemical research grade. S-1 was prepared from rabbit skeletal muscle (25Weeds A.G. Taylor R.S. Nature. 1975; 257: 54-56Crossref PubMed Scopus (931) Google Scholar), and the protein concentrations were determined from the absorbance at 280 nm as in my previous works (14Hiratsuka T. J. Biol. Chem. 1993; 268: 24742-24750Abstract Full Text PDF PubMed Google Scholar, 16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar). Experiments with protein samples were carried out in the following buffers: buffer A (25 mmHEPES, pH 8.0, 30 mm KCl, and 2 mmMgCl2), buffer B (50 mm Tris/Cl, pH 8.0, 0.5m KCl, and 5 mm CaCl2), and buffer C (50 mm Tris/Cl, pH 8.0, 0.5 m KCl, and 5 mm EDTA). Fluorescent labeling of S-1 with AD and BD was performed at 7 °C in buffer A in the dark. S-1 (2–2.5 mg/ml) was incubated with a 3-fold molar excess of the reagent over S-1. Because AD and BD were scarcely soluble in water, the reaction mixtures contained 0.3% N, N-dimethylformamide. The addition of 0.3% N, N-dimethylformamide had no effect on the K+-, Mg2+-, and Ca2+-ATPases of S-1. The reaction was stopped by the addition of dithiothreitol at a final concentration of 2 mm. The labeled S-1 was then separated from the unreacted reagent and the reaction products with dithiothreitol by passage through 1.5 × 9-cm columns of Sephadex G-50 equilibrated with 25 mm HEPES, pH 8.0, and 30 mm KCl. For measurements of ATPase activities, the samples were compared with a control subjected to the same conditions but excluding the reagent. The stoichiometry of the incorporated fluorophore/S-1 was determined from the absorption spectra in 25 mm HEPES, pH 8.0, and 30 mm KCl using extinction coefficients of 12,000 ± 300m−1 cm−1 at 382 nm for AD-S-1 and 12,300 ± 100 m−1 cm−1 at 392 nm for BD-S-1. These values were determined by comparing the absorption spectrum of the labeled S-1 with the amount of the thiol groups decreased. The labeled S-1, in which 0.62–0.98 thiol group/mol of S-1 had been labeled, was used for all experiments. The specific modifications of SH1 of S-1 with IAEDANS and IAA was done according to the procedures of Takashi et al. (26Takashi R. Duke J. Ue K. Morales M.F. Arch. Biochem. Biophys. 1976; 175: 279-283Crossref PubMed Scopus (70) Google Scholar) and Kunz et al. (27Kunz P.A. Walser J.T. Watterson J.G. Schaub M.C. FEBS Lett. 1977; 83: 137-140Crossref PubMed Scopus (16) Google Scholar), respectively. The resultant derivatives of S-1, in which 1–1.1 thiol groups/mol of S-1 had been modified, exhibited 5–10% of the K+-ATPase activity and 310–360% of the Ca2+-ATPase activity relative to those of control S-1. Thiol titrations were carried out to determine the extent of modification of thiol groups in S-1 by the method of Ellman's titration (28Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar), as described previously (14Hiratsuka T. J. Biol. Chem. 1993; 268: 24742-24750Abstract Full Text PDF PubMed Google Scholar). For unlabeled S-1, the amounts of thiol groups/mol of S-1 were 9.6 ± 0.15. These values were in excellent agreement with the values reported previously (29Schaub M.C. Watterson J.G. Walser J.T. Waser P.G. Biochemistry. 1978; 17: 246-253Crossref PubMed Scopus (17) Google Scholar, 30Hiratsuka T. Biochemistry. 1986; 25: 2101-2109Crossref PubMed Scopus (13) Google Scholar). Limited cleavage of the fluorescent derivatives of S-1 with trypsin and subsequent cleavage of the trypsinized samples at Asn-Gly bonds with hydroxyl amine (31Sutoh K. Biochemistry. 1981; 20: 3281-3285Crossref PubMed Scopus (34) Google Scholar) were performed as described previously (14Hiratsuka T. J. Biol. Chem. 1993; 268: 24742-24750Abstract Full Text PDF PubMed Google Scholar, 16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar). CNBr cleavage of fluorescent derivatives of S-1 were performed by the method of Elzinga and Collins (32Elzinga M. Collins J.H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4281-4284Crossref PubMed Scopus (99) Google Scholar). After CNBr cleavage, the samples were dialyzed against water with Spectropore 3 dialysis tubing (M r cut-off = 3,500). Finally, the samples were evaporated to dryness under reduced pressure at 30 °C. For SDS-PAGE, all the fragmented samples were dissolved with a solution containing 2% SDS, 10% 2-mercaptoethanol, 34% sucrose, and 0.1m sodium phosphate, pH 7.0. SDS-PAGE was carried out by the system of Weber and Osborn (33Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar) using 7.5 and 15% acrylamide gels, as described previously (16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar). To determine the distribution of fluorescence in the fluorescent derivatives of S-1, the S-1 samples were subjected to SDS-PAGE, and then relative amounts of fluorescence extracted from peptide bands were measured as described previously (34Hiratsuka T. J. Biol. Chem. 1989; 264: 18188-18194Abstract Full Text PDF PubMed Google Scholar). The Ca2+- and K+-ATPase activities were measured at 25 °C in 1 mm ATP in buffers B and C, respectively. Piliberated was determined by the method of Fiske and SubbaRow (35Fiske C.H. SubbaRow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). The ATPase activities of the unlabeled S-1 in μmol of Pi/min/mg were: K+-ATPase, 10; Ca2+-ATPase, 1.2. All spectral measurements of the thiol-modified derivatives of S-1 were performed in buffer A. Absorption spectra were measured at room temperature with a Shimadzu MPS-2000 spectrophotometer. Corrected fluorescence emission spectra were recorded at 25 °C in a thermostatted Hitachi fluorescence spectrophotometer (model MPF-4). All fluorescence measurements with S-1 samples (0.4–0.75 μm) were performed in the presence and absence of ligands (1 mm). Because fluorescence measurements were completed within 15 min, the data in the presence of ATP were regarded as those obtained during the steady state of ATP hydrolysis. Excitation wavelength was 390 nm. The slit widths on excitation and emission monochromators were 5 nm. For the measurements of the labeling rates of S-1 with AD and BD, increases in fluorescence at 500 nm were monitored at 7 °C. The freshly prepared KI solution was used. The stock solution (5 m) contained 10−4m sodium thiosulfate to prevent oxidation of I−. All measurements were carried out at 25 °C with excitation and emission lights of 390 and 500 nm, respectively. Data were shown as Stern-Volmer plots to obtain the quenching constant,K SV (36Stern O. Volmer M. Phys. Z. 1919; 20: 183Google Scholar). Concentrations of thiol-modified S-1 were determined by the biuret method, standardized against control S-1. Control experiments showed that the modification of S-1 did not interfere with protein determinations by the biuret methods. S-1 was assumed to have an M r of 120,000 (25Weeds A.G. Taylor R.S. Nature. 1975; 257: 54-56Crossref PubMed Scopus (931) Google Scholar). Alkylation of S-1 with IAA and IAEDANS invariably results in the modification of only SH1 (20Reisler E. Methods Enzymol. 1982; 85: 84-93Crossref PubMed Scopus (82) Google Scholar, 26Takashi R. Duke J. Ue K. Morales M.F. Arch. Biochem. Biophys. 1976; 175: 279-283Crossref PubMed Scopus (70) Google Scholar, 27Kunz P.A. Walser J.T. Watterson J.G. Schaub M.C. FEBS Lett. 1977; 83: 137-140Crossref PubMed Scopus (16) Google Scholar). Although chemical properties of BD had scarcely been studied, the reagent was expected to be useful as a specific fluorescent label for SH1 of S-1. This is because the reactivity of the functional group of BD, the bromoacetyl group, is similar to those of functional groups of IAA and IAEDANS (Scheme FS1). On the other hand, the structure of the fluorophore moiety of BD, the 6-acyl-2-dimethylaminonaphthalene group, is the same as those of another thiol reagent AD and the noncovalent probe prodan (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar, 22Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar). Thus BD can be expected to show fluorescence that is extremely sensitive to solvent polarity as well as prodan and AD (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar, 22Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar, 23Marriott G. Zechel K. Jovin T.M. Biochemistry. 1988; 27: 6214-6220Crossref PubMed Scopus (45) Google Scholar, 24Hiratsuka T. Biochemistry. 1998; 37: 7167-7176Crossref PubMed Scopus (15) Google Scholar). The absorption spectra of BD and AD derivatives of S-1 exhibited a maximum at 278 nm together with those at 382 nm (AD-S-1) and 392 nm (BD-S-1) (Fig.1). The maxima around 380–390 nm are associated entirely with the 6-acyl-2-dimethylaminonaphthalene moiety of AD and BD (21Weber G. Farris F.J. Biochemistry. 1979; 18: 3075-3078Crossref PubMed Scopus (724) Google Scholar, 22Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar). Upon excitation at 390 nm, the S-1 derivatives fluoresced strongly with a single maximum at 502 nm (AD-S-1) and 508 nm (BD-S-1) (Fig. 1). On the other hand, the AD adduct of 2-mercapto-ethanol fluoresced at 535 nm (Fig. 1, inset). However, the fluorescence intensity of AD itself was less than 4% relative to that of the fluorescent adduct. Similar results were obtained in the case of the reaction of 2-mercaptoethanol with BD (not shown). This indicates that the formation of the fluorescent derivatives of S-1 can be conveniently monitored by measurements of an increase in fluorescence that is associated with fluorophores attached to S-1 (see below). Fig. 2 shows the time courses of labeling of S-1 with AD, which occurred at 7 °C with a 3-fold molar excess of AD over S-1. They were followed by measurements of the amount of labeled thiol groups and fluorescence increase at 500 nm. The time courses obtained by both measurements agreed well. The data indicate that the labeling of S-1 is relatively rapid for the first 30 min and much slower thereafter. The labeling reaction was completed in 1 h, resulting in the incorporation of 0.8–1.0 AD group/mol of S-1. It should be noted that the existence of 0.9 mol of the first-labeled thiol group is indicated by extraporation of the slow labeling to zero time. Fig. 2 (inset) shows the relationship between the percentage of remaining ATPase activities of AD-S-1 and the number of the labeled thiol groups/mol of S-1. The K+-ATPase activity was decreased linearly with increasing amount of the labeled thiol group. The decrease of 1 mol of thiol group led to a 70% loss of activity. On the other hand, the Ca2+-ATPase activity was scarcely affected by the labeling. Fig. 3 A shows the time course of the labeling of S-1 with BD, which has occurred under the same condition as that of the labeling with AD (Fig. 2). The rate of the labeling with BD was much faster than that of the labeling with AD. 1 mol of thiol group/mol of S-1 was labeled within 5 min. The existence of 1 mol of the first-labeled thiol group/mol of S-1 was indicated by extraporation of the slow labeling to zero time. However, unlike the labeling with AD, another thiol group of S-1 was also labeled within 25 min (see below). The effect of BD on the K+- and Ca2+-ATPase activities of S-1 was also followed. Fig. 3 B shows that as thiol groups are labeled with BD the Ca2+-ATPase activity increases, while the K+-ATPase activity decreases. When 1.2 thiol group/mol of S-1 was labeled, the Ca2+-ATPase activity was increased to 220% of the original value. Thereafter, the activity began to plateau and then decreased. The linear relationship between the Ca2+-ATPase activity and the loss of K+-ATPase activity was observed (Fig. 3 B,inset). The enzymatic properties of fluorescent derivatives of S-1 described above are indicative of the labeling of SH1 with BD and SH2 with AD (12Phan B.C. Peyser Y.M. Reisler E. Muhlrad A. Eur. J. Biochem. 1997; 243: 636-642Crossref PubMed Scopus (40) Google Scholar, 13Ajtai K. Burghardt T.P. Biochemistry. 1989; 28: 2204-2210Crossref PubMed Scopus (34) Google Scholar, 14Hiratsuka T. J. Biol. Chem. 1993; 268: 24742-24750Abstract Full Text PDF PubMed Google Scholar, 15Hiratsuka T. Biochemistry. 1988; 27: 4110-4114Crossref PubMed Scopus (10) Google Scholar, 16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar, 17Rajasekharan K.N. Mayadevi M. Burke M. J. Biol. Chem. 1989; 264: 10810-10819Abstract Full Text PDF PubMed Google Scholar, 18Kasprazk A.A. Chaussepied P. Morales M.F. Biochemistry. 1989; 28: 9230-9238Crossref PubMed Scopus (19) Google Scholar, 19Xing J. Cheung H.C. Biochemistry. 1995; 34: 6475-6487Crossref PubMed Scopus (22) Google Scholar, 20Reisler E. Methods Enzymol. 1982; 85: 84-93Crossref PubMed Scopus (82) Google Scholar,26Takashi R. Duke J. Ue K. Morales M.F. Arch. Biochem. Biophys. 1976; 175: 279-283Crossref PubMed Scopus (70) Google Scholar, 27Kunz P.A. Walser J.T. Watterson J.G. Schaub M.C. FEBS Lett. 1977; 83: 137-140Crossref PubMed Scopus (16) Google Scholar). To ascertain this point, the locations of AD and BD attached to S-1 were defined by fragmenting the S-1 samples with both trypsin and CNBr (16Hiratsuka T. J. Biol. Chem. 1992; 267: 14941-14948Abstract Full Text PDF PubMed Google Scholar, 32Elzinga M. Collins J.H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4281-4284Crossref PubMed Scopus (99) Google Scholar). All the samples were run on SDS-PAGE, followed by the examination under UV illumination. The S-1 derivative where SH1 had been fluorescently labeled with IAEDANS (26Takashi R. Duke J. Ue K. Morales M.F. Arch. Biochem. Biophys. 1976; 175: 279-283Crossref PubMed Scopus (70) Google Scholar) was also used as a standard. As shown in Fig. 4 A, all fluorescent labels are predominantly localized in the heavy chain of S-1. However, some labeling of light chain-1 was found with BD. Relative amounts of fluorescence extracted from peptides bands of heavy chain and the combined light chains were 93 and 7% (IAEDANS-S-1), 78 and 22% (BD-S-1), and 91 and 9% (AD-S-1), respectively. For the tryptic digests, fluorescence was found only on the 20-kDa band (Fig.4 B). Upon CNBr cleavage for the samples, fluorescence was observed on the 10-kDa segment (Fig. 4 C), showing that only thiol groups that had been labeled were either SH1 or SH2 for both the BD and AD derivatives of S-1 (32Elzinga M. Collins J.H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4281-4284Crossref PubMed Scopus (99) Google Scholar). It is well established that hydroxylamine cleaves the 20-kDa tryptic peptide of S-1 at the single Asn-Gly bond to produce a C-terminal 13-kDa peptide containing SH1 and a 7-kDa peptide containing SH2 (31Sutoh K. Biochemistry. 1981; 20: 3281-3285Crossref PubMed Scopus (34) Google Scholar). However, the usual demonstration using this hydroxylamine cut is not possible with AD and BD because of the instability of the labels to the extreme pH and temperature conditions of the hydroxylamine treatment. I found that fluorescence of the samples of AD-S-1 and BD-S-1 disappeared by the treatment, although that of the sample of IAEDANS-S-1 remained unaltered. Because of this complication I tried an indirect approach where IAA-S-1 was further labeled with BD and AD. As summarized in Table I, IAA-S-1 was further incubated with BD and AD for 3 and 30 min, respectively. For control S-1, the amount of thiol groups labeled with BD was 1.05 mol/mol of S-1. However, for IAA-S-1, the amount was diminished to be only 0.19 mol/mol of S-1. Thus 0.86 thiol group/mol of S-1 (82% of the control value) was unlabeled with BD after treatment with IAA. This suggests that BD labels SH1 predominantly together with a slight labeling of light chain-1 (Figs. 3 A and 4 A). On the other hand, the labeling with AD was scarcely affected by the pretreatment with IAA. The amounts of labeled thiol groups were 0.90 and 0.82 mol/mol of S-1 for control S-1 and IAA-S-1, respectively. This indicates that AD can specifically label SH2. SDS-PAGE patterns for the samples of AD derivatives of S-1 and IAA-S-1 confirmed this result (Fig. 4 D). In the case of labeling with AD, no difference in fluorescence intensities between S-1 and IAA-S-1 was observed on both bands of the heavy chain and the 20-kDa tryptic peptide. On the other hand, fluorescence was scarcely observed on the bands of IAA-S-1 in the case of labeling with IAEDANS.Table IFluorescent labeling of IAA-S-1 with AD and BDSampleaIAA-S-1 in which 1.1 thiol groups/mol of S-1 had been modified was used.BDbIncubation times were 30 and 3 min for AD and BD, respectively.ADbIncubation times were 30 and 3 min for AD and BD, respectively.Amount of SH labeledAmount of SH protected with IAAmol/mol of S-1mol/mol of S-1Control S-1+−1.05IAA-S-1+−0.190.86Control S-1−+0.90IAA-S-1−+0.820.08Control S-1 and IAA-S-1 (2 mg/ml) were incubated at 7 °C in buffer A in the presence (+) and absence (−) of a 3-fold molar excess of the reagent over S-1, followed by passage through a column of Sephadex G-50 and then subjected to the assays of thiol contents as described under “Experimental Procedures.”a IAA-S-1 in which 1.1 thiol groups/mol of S-1 had been modified was used.b Incubation times were 30 and 3 min for AD and BD, respectively. Open table in a new tab Control S-1 and IAA-S-1 (2 mg/ml) were incubated at 7 °C in buffer A in the presence (+) and absence (−) of a 3-fold molar excess of the reagent over S-1, followed by passage through a column of Sephadex G-50 and then subjected to the assays of thiol contents as described under “Experimental Procedures.” Taking into account the enzymatic" @default.
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• W2061340145 title "ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence" @default.
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