Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum.
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.
Taylor
K A
KA
Department of Anatomy, Duke University Medical Center, Durham, North Carolina 27710.
Mullner
N
N
Pikula
S
S
Dux
L
L
Peracchia
C
C
Varga
S
S
Martonosi
A
A
D000818:Animals; D000252:Calcium-Transporting ATPases; D003460:Crystallization; D003902:Detergents; D005614:Freeze Fracturing; D008854:Microscopy, Electron; D008958:Models, Molecular; D010743:Phospholipids; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility; D013501:Surface-Active Agents; D014961:X-Ray Diffraction
D003902:Detergents; D010743:Phospholipids; D013501:Surface-Active Agents; D000252:Calcium-Transporting ATPases