Stabilization and crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum.
Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases; D003460:Crystallization; D003902:Detergents / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005990:Glycerol / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019810:Sodium Azide; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
D001386:Azides; D003902:Detergents; D013501:Surface-Active Agents; D014638:Vanadates; D004533:Egtazic Acid; D007611:Aprotinin; D019810:Sodium Azide; D000252:Calcium-Transporting ATPases; D005990:Glycerol; D002118:Calcium; D004229:Dithiothreitol