Correlation of structure and function in the Ca2+-ATPase of sarcoplasmic reticulum: a Fourier transform infrared spectroscopy (FTIR) study on the effects of dimethyl sulfoxide and urea.
The effect of dimethyl sulfoxide (DMSO) on the structure of sarcoplasmic reticulum was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopy. Exposure of sarcoplasmic reticulum vesicles to 35% DMSO (v/v) at 2 degrees C for several hours in a D2O medium produced no significant change in the phospholipid and protein Amide I regions of the FTIR spectra, but the intensity of the Amide II band decreased, presumably due to proton/deuterium exchange. At 40% to 60% DMSO concentration a shoulder appeared in the FTIR spectra at 1630 cm-1, that is attributed to the formation of new beta or random coil structures; irreversible loss of ATPase activity accompanied this change. At 70% DMSO concentration the intensity of the main Amide I band at 1639 cm-1 decreased and a new band appeared at 1622 cm-1, together with a shoulder at 1682 cm-1. These changes indicate an abrupt shift in the conformational equilibrium of Ca2+-ATPase from alpha to beta structure or to a new structure characterized by weaker hydrogen bonding. Decrease of ionization of aspartate and glutamate carboxyl groups in the presence of DMSO may also contribute to the change in intensity at 1622 cm-1. The changes were partially reversed upon removal of DMSO. Exposure of sarcoplasmic reticulum vesicles to 1.5 kbar pressure for 1 h at 2 degrees C in an EGTA-containing (low Ca2+) medium causes irreversible loss of ATPase activity, with the appearance of new beta structure, and abolition of the Ca2+-induced fluorescence response of FITC covalently bound to the Ca2+-ATPase; DMSO (35%) stabilized the Ca2+-ATPase against pressure-induced changes in structure and enzymatic activity, while urea (0.8 M) had the opposite effect.
Buchet
R
R
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Jona
I
I
Martonosi
A
A
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005583:Fourier Analysis; D011312:Pressure; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship / Q000187:drug effects; D013861:Thiocyanates; D014508:Urea / Q000494:pharmacology*
D005452:Fluoresceins; D013861:Thiocyanates; D014508:Urea; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D004121:Dimethyl Sulfoxide