Pressure effects on sarcoplasmic reticulum.
The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800-2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+-ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure.
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002463:Cell Membrane Permeability; D003460:Crystallization; D004533:Egtazic Acid / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008433:Mathematics; D011189:Potassium Chloride / Q000494:pharmacology; D011312:Pressure; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013861:Thiocyanates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D005452:Fluoresceins; D008301:Maleimides; D013861:Thiocyanates; D014639:Vanadium; D014638:Vanadates; D004533:Egtazic Acid; D011189:Potassium Chloride; C003944:N-(3-pyrene)maleimide; D009597:4-Nitrophenylphosphatase; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate