title: The effect of high pressure on the conformation, interactions and activity of the Ca(2+)-ATPase of sarcoplasmic reticulum.
abstract: High pressure (100-150 MPa) increases the intensity and polarization of fluorescence of FITC-labeled Ca(2+)-ATPase in a medium containing 0.1 mM Ca2+, suggesting a reversible pressure-induced transition from the E1 into an E2-like state with dissociation of ATPase oligomers. Under similar conditions but using unlabeled sarcoplasmic reticulum vesicles, high pressure caused the reversible release of Ca2+ from the high-affinity Ca2+ sites of Ca(2+)-ATPase, as indicated by changes in the fluorescence of the Ca2+ indicator, Fluo-3; this was accompanied by reversible inhibition of the Ca(2+)-stimulated ATPase activity measured in a coupled enzyme system of pyruvate kinase and lactate dehydrogenase, and by redistribution of Prodan in the lipid phase of the membrane, as shown by marked changes in its fluorescence emission characteristics. In a Ca(2+)-free medium where the equilibrium favors the E2 conformation of Ca(2+)-ATPase the fluorescence intensity of FITC-ATPase was not affected or only slightly reduced by high pressure. The enhancement of TNP-AMP fluorescence by 100 mM inorganic phosphate in the presence of EGTA and 20% dimethylsulfoxide was essentially unaffected by 150 MPa pressure at pH 6.0 and was only slightly reduced at pH 8.0. As the enhancement of TNP-AMP fluorescence by Pi is associated with the Mg(2+)-dependent phosphorylation of the enzyme and the formation of Mg.E2-P intermediate, it appears that the reactions of Ca(2+)-ATPase associated with the E2 state are relatively insensitive to high pressure. These observations suggest that high pressure stabilizes the enzyme in an E2-like state characterized by low reactivity with ATP and Ca2+ and high reactivity with Pi. The transition from the E1 to the E2-like state involves a decrease in the effective volume of Ca(2+)-ATPase.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
authors.forename: I
authors.initials: I
authors.lastname: Jona
authors.forename: A
authors.initials: A
authors.lastname: Martonosi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011312:Pressure; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis; D014638:Vanadates / Q000494:pharmacology
chemical_list: D014638:Vanadates; D004533:Egtazic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium