Rapid reconstitution and characterization of highly-efficient sarcoplasmic reticulum Ca pump.
The Ca pump was reconstituted from the purified sarcoplasmic reticulum ATPase and excess soybean phospholipids by the freeze-thaw sonication procedure in the presence of cholate. In the absence of Ca precipitating agents, the reconstituted proteoliposomes accumulated Ca2+ at an initial rate of up to 0.7 mumol/mg per min at 25 degrees C, and a value of 1.54 was obtained for the coupling ratio between Ca uptake and Ca2+-dependent ATPase activities. The proteoliposomes were mainly unilamellar vesicles but were heterogeneous with respect to their size. When reconstituted at a lipid/protein ratio of 40, proteoliposomes had a buoyant density of about 1.04 and their average internal volume was 1.4-1.6 microliters/mg of phospholipids. More than 95% of the ATPase was incorporated randomly into these proteoliposomes and the fraction of proteoliposomes that represented about 50% of the total intravesicular isotope space contained right-side-out oriented enzyme. 86Rb efflux from the 86Rb-loaded proteoliposomes was found to be slow even at 25 degrees C. Therefore, the proteoliposomes prepared by the present simple method should be useful for the study of the side-specific interaction of ions such as alkali metal cations with the sarcoplasmic reticulum Ca pump.
Wakabayashi
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Shigekawa
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011197:Potassium Radioisotopes / Q000378:metabolism; D011817:Rabbits; D011868:Radioisotopes; D012413:Rubidium / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002793:Cholic Acids; D007473:Ion Channels; D008081:Liposomes; D010743:Phospholipids; D011197:Potassium Radioisotopes; D011868:Radioisotopes; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D019826:Cholic Acid; D012413:Rubidium; D002118:Calcium