Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum

• Published in: The Journal of biological chemistry Vol. 263; no. 11; pp. 5287 - 5294
• Main Authors: Taylor, K A, Mullner, N, Pikula, S, Dux, L, Peracchia, C, Varga, S, Martonosi, A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.04.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium-Transporting ATPases; Crystallization; Detergents; Enzymes and enzyme inhibitors; Freeze Fracturing; Fundamental and applied biological sciences. Psychology; Hydrolases; Microscopy, Electron; Models, Molecular; Phospholipids; Rabbits; Sarcoplasmic Reticulum - enzymology; Solubility; Surface-Active Agents; X-Ray Diffraction; Ca-ATPase; Detergent; Transmission electron microscopy; Calcium; Enzyme; Sarcoplasmic reticulum; Ion transport; Freeze etching; Kinetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)60713-6

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.