Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.
Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.
Lüdi
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Hasselbach
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D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D010743:Phospholipids / Q000032:analysis; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010743:Phospholipids; D000252:Calcium-Transporting ATPases