[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1
Tóm tắt
The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0°C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-3H]ADP.Vi shifts both peaks slightly more upfield without chaning their widths or relative areas.
Tài liệu tham khảo
Al-Rawi, J.M.A., Bloxsidge, J.P., O'Brien, C., Caddy, D.E., Elridge, J.A., Jones, J.R. and Evans, E.A. (1974) J. Chem. Soc. Perkin Trans. 2, 1635–1639.
Bagshaw, C.R. and Trentham, D.R. (1974) Biochem. J., 141, 331–349.
Botts, J., Thomason, J.F. and Morales, M.F. (1989) Proc. Natl. Acad. Sci. USA, 86, 2204–2208.
Cremo, C.R., Grammer, J.C. and Yount, R.G. (1989) J. Biol. Chem., 264, 6608–6611.
Dwek, R.A. (1973) Nuclear Magnetic Resonance in Biochemistry, Clarendon, Oxford, pp. 48–54.
Eckstein, F. and Goumet, M. (1978) In Nucleic Acid Chemistry, Vol. 2 (Eds., Townsend, L.B. and Tipson, R.S.) Wiley, New York, pp. 861–864.
Elvidge, J.A., Jones, J.R., O'Brien, C., Evans, E.A. and Sheppard, H.C. (1973) J. Chem. Soc., Perkin Trans. 2, 2138–2141.
Evans, E.A., Sheppard, H.C., Turner, J.C. and Warrell, D.C. (1974) J. Label. Compounds Radiopharm., 10, 569–587.
Goodno, C.C. (1979) Proc. Natl. Acad. Sci. USA, 76, 2620–2624.
Highsmith, S. (1976) J. Biol. Chem., 251, 6170–6172.
Highsmith, S. and Murphy, A.J. (1992) Biochemistry, 31, 385–389.
Highsmith, S., Akasaka, K., Konrad, M., Goody, R., Holmes, K., Wade-Jardetzky, N. and Jardetzky, O. (1979) Biochemistry, 18, 4238–4244.
Hoard, D.E. and Ott, D.G. (1965) J. Am. Chem. Soc., 87, 1785–1788.
Jardetzky, O. and Roberts, G.C.K. (1981) NMR in Molecular Biology, Academic Press, New York, pp. 379–412.
Kasprzak, A.A., Chaussepied, P. and Morales, M.F. (1989) Biochemistry, 28, 9230–9238.
Kendrick-Jones, J., Lehman, W. and Szent-Gyoryi, A.G. (1970) Methods Enzymol., 11, 199–203.
Lin, S.H. and Cheung, H.C. (1991) Biochemistry, 30, 4317–4322.
Mahmood, R., Elzinga, M. and Yount, R.G. (1989) Biochemistry, 28, 3989–3995.
Montgomery, J.A. and Hewson, K. (1964) J. Heterocyclic Chem., 1, 213–214.
Morita, F. (1967) J. Biol. Chem., 242, 4501–4506.
Murphy, A.J. (1974) Arch. Biochem. Biophys., 163, 290–296.
Nair, V. and Richardson, S.G. (1982) Synthesis, 670–672.
Newmark, R.D., Un, S., Williams, P.G., Carson, P.L., Morimoto, H. and Klein, M.P. (1990) Proc. Natl. Acad. Sci. USA, 87, 583–587.
Okamoto, Y. and Yount, R.G. (1985) Proc. Natl. Acad. Sci. USA, 82, 155–159.
Papp, S., Eden, D. and Highsmith, S. (1992) Biochim. Biophys. Acta, 1159, 267–273.
Pate, E., Nakamaye, K.L., Franks-Skiba, K., Yount, R.G. and Cooke, R. (1991) Biophys. J., 59, 598–605.
Peyser, Y.M., Muhlrad, A. and Werber, M.M. (1990) FEBS Lett., 259, 346–348.
Prince, H.P., Trayer, H.R., Henry, G.D., Trayer, I.P., Dalgarno, D.C., Levine, B.A., Cary, P.D. and Turner, C. (1981) Eur. J. Biochem., 121, 213–219.
Robins, M.J. and Uznanski, B. (1986) In Nucleic Acid Chemistry, Vol. 3 (Eds., Townsend, L.B. and Tipson, R.S.) Wiley, New York, pp. 144–148.
Rosenfield, S.S. and Taylor, E.W. (1984) J. Biol. Chem., 259, 11920–11929.
Shriver, J.W. and Sykes, B.D. (1981) Biochemistry, 20, 2004–2012.
Sutoh, K. (1987) Biochemistry, 26, 7648–7654.
Taylor, E.D. (1979) CRC Crit. Rev. Biochem., 6, 103–106.
Tokunaga, M., Sutoh, K., Toyoshima, C. and Wakabayashi, T. (1987) Nature (London), 329, 635–638.
Toyoshima, Y.Y., Kron, S.J., McNally, E.M., Niebling, K.R., Toyoshima, C. and Spudich, J.A. (1987) Nature (London), 328, 536–539.
Wagner, P.D. and Giniger, E. (1981) Nature (London), 292, 560–562.
Walker, J.E., Seraste, M., Runswick, M.J. and Gay, N.J. (1982) EMBO J., 1, 945–951.
Weeds, A.G. and Taylor, R.S. (1975) Nature (London), 257, 54–56.
Werber, M.M., Szent-Gyorgyi, A.G. and Fasman, G.D. (1972) Biochemistry, 11, 2872–2883.
Yamamoto, K. (1990) Biochemistry, 29, 844–848.