| 2965700 |
Taylor KA, Mullner N, Pikula S, Dux L, Peracchia C, Varga S, Martonosi A: Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum. J Biol Chem. 1988 Apr 15;263(11):5287-94.
Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 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. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations. |
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