期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1974
卷号：71
期号：3
页码：622-626
DOI：10.1073/pnas.71.3.622
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A (Mg2+ + Ca2+)ATPase (ATP phosphohydrolase, EC 3.6.1.3 ) has been purified from sarcoplasmic reticulum using a single step centrifugation procedure. The preparation is >95% pure by weight and contains only 25-30% of the lipid associated with the enzyme in native sarcoplasmic reticulum. The purified enzyme is unable to accumulate Ca2+. Using a sedimentation-substitution technique, >98% of the lipid associated with the purified enzyme can be replaced by dioleoyl lecithin without grossly affecting the ATPase activity of the enzyme. The Ca2+ pump can be restored to this dioleoyl lecithin-substituted enzyme by addition of excess sarcoplasmic reticulum lipids in the presence of cholate. Removal of the cholate by dialysis generates a system which accumulates Ca2+ at a rate and to a level comparable to native sarcoplasmic reticulum. Significant reconstitution of the Ca2+ pump can also be achieved using excess dioleoyl lecithin, but since the full expression of the capacity to accumulate Ca2+ requires the presence of oxalate, these vesicles would appear to be more leaky than those reconstituted with an excess of sarcoplasmic reticulum lipids. Of about 90 lipid molecules which are associated with one molecule of ATPase in native sarcoplasmic reticulum, an average of less than one lipid molecule remains in these reconstituted systems. We have therefore achieved a fully functional Ca2+ pump containing essentially a single protein and exogenous lipid.
关键词：(Mg2+ + Ca2+)ATPase ; sarcoplasmic reticulum ; active transport ; membrane protein purification ; lipid substitution
