Methods of reconstitution of the purified sarcoplasmic reticulum (Ca(2+)-Mg2+)-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles.
A detailed methodology is presented of two reconstitution protocols for the (Ca(2+)-Mg2+)-ATPase from rabbit skeletal muscle, using the detergent potassium cholate. Method A was shown to produce fully fragmented membranes of definable lipid to protein ratios, which were unable to take up calcium upon hydrolysis of ATP. This protocol was shown to produce a homologous population of membranes with respect to their lipid and protein composition at lipid to protein ratios up to 900:1 (mol/mol). Method B produced vesicles only of high lipid to protein ratios (3000:1), which have the ability to accumulate calcium on addition of ATP. Calcium accumulation and ATP hydrolysis for the ATPase reconstituted into different fatty acyl chain length phospholipids were also studied.
Michelangeli
F
F
Istituto di Patologia Generale, Università di Padova, Italy.
Munkonge
F M
FM
D000818:Animals; D001647:Bile Acids and Salts; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents; D005260:Female; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008722:Methods; D009124:Muscle Proteins / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D001647:Bile Acids and Salts; D003902:Detergents; D008055:Lipids; D009124:Muscle Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium