Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation
著者
山崎, 和生
(Yamasaki, Kazuo)
Daiho, T
Suzuki, H
上位タイトル
Journal of Biological Chemistry
Vol.277,
No.16
(2002.
4)
,p.13615-
13619
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.
注記
American Society for Biochemistry and Molecular Biology, Yamasaki, Kazuo ; Daiho, Takashi ; Suzuki, Hiroshi, Journal of Biological Chemistry, 277(16), 2002, 13615-13619
