A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities.
A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D000252:Calcium-Transporting ATPases; D002118:Calcium