Abstract
Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.
Highlights
Sarcoplasmic reticulum vesicleswere isolated and their protein composition and enzymatic activity were assayed according to Varga et al (1986)
By systemoctaethyleneglycoldodecyletherat a ratioof 2:l. atically testing several hundred conditions, we found that the Similar Ca2+-inducecdrystalline arrays were obtained Ca2+-modulatedATPase activity is preserved for 1-2 months with the purified or delipidated Ca2+-ATPase of sar- at 2 “C under nitrogen in a crystallization medium of 0.1 M coplasmic reticulum but at lower detergent:protreain- KC1,lO mM K-MOPS,’ pH 6.0, 3 mM MgCl, 3 mM NaN3, 5 tios
As a first step of the Ca2+-inducedEl-type crystals of the Ca2+-ATPase(Dux in that direction we report the formation of microcrystals of et al, 1985)
Summary
Sarcoplasmic reticulum vesicleswere isolated and their protein composition and enzymatic activity were assayed according to Varga et al (1986). Freeze-fracture electron microscopy was performed as described by Peracchia et al (1984), the negative staining according to Dux and Martonosi (1984), and the electron microscope analysis of sectioned material essentially according to Jilka et al (1975). The purified Caz+-ATPasewas prepared by the method of Meissner et al (1973) and delipidated with deoxycholate according to Dean and Tanford (1978). The phospholipid content and composition were analyzed by solvent extraction and thin-layer chromatography as described by Sarzala and Michalak (1978)
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