Pressure effects on sacroplasmic reticulum: a Fourier transform infrared spectroscpic study

Abstract

The Ca 2+-ATPase of sacroplasmic reticulum is irreversibly inactivated by exposure to 1.5–2.0 kbar pressure for 30–60 min in a Ca 2+-free medium; mono- or decavanadate (5 mM) or to a lesser extent Ca 2+ (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 2H 2O medium. The inactivation of the Ca 2+-ATPase at 1.5–2.0 kbar pressure in a Ca 2+-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 β sheet. The frequency of the peak of the Amide 1 band shifted from about 1648 cm −1 at atmospheric pressure to 1642 cm −1 at ≅ 12.5 kbar pressure, suggesting a decrease in α helix, and an increase in β 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 Ca 2+ (2–20 mM), protected the Ca 2+-ATPase against pressure-induced changes both in the Amide I and Amide II regions of the spectrum, together with the protection of ATPase activity. These observations establish a correlation between the conformation of the Ca 2+-ATPase and its sensitivity to pressure. The involvement of the ATP binding domain of the Ca 2+-ATPase in the pressure-induced structural changes is suggested by the decreased polarization of fluorescence of fluorescein 5′-isothiocyanate covalently attached to the enzyme.