Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation.
Conditions were developed in the absence of Ca(2+) for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C(12)E(8))-solubilized sarcoplasmic reticulum Ca(2+)-ATPase with tightly bound Mg(2+) and F(-), an analog for the phosphoenzyme intermediate without bound Ca(2+). The Ca(2+)-ATPase activity to monitor denaturation was assessed after treatment with 20 mm Ca(2+) to release tightly bound Mg(2+)/F(-). The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg(2+)/F(-)-bound Ca(2+)-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4 degrees C even without exogenously added phospholipid and at a high C(12)E(8)/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mm Ca(2+), showing that tightly bound Mg(2+)/F(-) was not released during the long term incubation. The perfect stability was attained with or without 0.1 mm dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mm dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca(2+)-ATPase molecule induced by tight binding of Mg(2+)/F(-). The present study thus reveals an essential property of the Mg(2+)/F(-)/Ca(2+)-ATPase complex, which will likely provide clues to understanding structure of the Ca(2+)-released form of phosphoenzyme intermediate at an atomic level.
Yamasaki
Kazuo
K
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
Daiho
Takashi
T
Suzuki
Hiroshi
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D005459:Fluorides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008274:Magnesium / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013997:Time Factors
D003902:Detergents; D004220:Disulfides; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides; D002118:Calcium; D004229:Dithiothreitol