Buchet R, Carrier D, Wong P T, Jona I, Martonosi A
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Biochim Biophys Acta. 1990 Mar 30;1023(1):107-18. doi: 10.1016/0005-2736(90)90015-g.
The Ca2(+)-ATPase of sarcoplasmic reticulum is irreversibly inactivated by exposure to 1.5-2.0 kbar pressure for 30-60 min in a Ca2(+)-free medium; mono- or decavanadate (5 mM) or to a lesser extent Ca2+ (2-20 mM) protect against inactivation (Varga et al. (1986) J. Biol. Chem. 261, 13943-13956). The structural basis of these effects was analyzed by FTIR spectroscopy of sarcoplasmic reticulum in 2H2O medium. The inactivation of the Ca2(+)-ATPase at 1.5-2.0 kbar pressure in a Ca2(+)-free medium was accompanied by changes in the Amide II region of the spectrum (1550 cm-1), that are consistent with increased hydrogen-deuterium (H-2H) exchange, and by the enhancement of a band at 1630 cm-1 in the Amide I region, that is attributed to an increase in beta sheet. The frequency of the peak of the Amide I band shifted from about 1648 cm-1 at atmospheric pressure to 1642 cm-1 at approximately equal to 12.5 kbar pressure, suggesting a decrease in alpha helix, and an increase in beta and/or random coil structures. Upon releasing the pressure, the shift of the Amide I band was partially reversed. Vanadate (5 mM), and to a lesser extent Ca2+ (2-20 mM), protected the Ca2(+)-ATPase against pressure-induced changes both in the Amide I and Amide II regions of the spectrum, together with protection of ATPase activity. These observations establish a correlation between the conformation of the Ca2(+)-ATPase and its sensitivity to pressure. The involvement of the ATP binding domain of the Ca2(+)-ATPase in the pressure-induced structural changes is suggested by the decreased polarization of fluorescence of fluorescein 5'-isothiocyanate covalently attached to the enzyme.
在无钙介质中，肌浆网的Ca2(+)-ATP酶暴露于1.5 - 2.0千巴压力30 - 60分钟会被不可逆地灭活；单钒酸盐或十钒酸盐（5 mM）或在较小程度上Ca2+（2 - 20 mM）可防止其灭活（瓦尔加等人（1986年）《生物化学杂志》261卷，13943 - 13956页）。通过在2H2O介质中对肌浆网进行傅里叶变换红外光谱分析，研究了这些效应的结构基础。在无钙介质中，1.5 - 2.0千巴压力下Ca2(+)-ATP酶的失活伴随着光谱酰胺II区（1550 cm-1）的变化，这与氢-氘（H-2H）交换增加一致，同时酰胺I区1630 cm-1处的谱带增强，这归因于β折叠增加。酰胺I带峰的频率从大气压下的约1648 cm-1移至约12.5千巴压力下的1642 cm-1，表明α螺旋减少，β折叠和/或无规卷曲结构增加。压力释放后，酰胺I带的位移部分逆转。钒酸盐（5 mM）以及在较小程度上Ca2+（2 - 20 mM）保护Ca2(+)-ATP酶免受光谱酰胺I区和酰胺II区压力诱导的变化，同时保护ATP酶活性。这些观察结果建立了Ca2(+)-ATP酶构象与其对压力敏感性之间的相关性。与酶共价连接的异硫氰酸荧光素5'-异硫氰酸酯荧光偏振度降低，表明Ca2(+)-ATP酶的ATP结合结构域参与了压力诱导的结构变化。
