Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.
Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Pikułla, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.
Pikuła
S
S
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Wrzosek
A
A
Famulski
K S
KS
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood* / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology*; D004795:Enzyme Stability; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility; D013552:Swine; D014961:X-Ray Diffraction
D003902:Detergents; D010743:Phospholipids; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium