Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase.
The Ca2+-dependent ATPase of sarcoplasmic reticulum after solubilization with deoxycholate and removal of lipid by gel chromatography exists as a mixture of monomer, dimer, and smaller amounts of higher molecular weight aggregates. The binding capcity of deoxycholate by monomeric and oligomeric forms of the ATPase is 0.3 g/g of protein at pH 8 and ionic strength 0.11. Examination in the analytical ultracentrifuge results in estimates of protein molecular weight of monomer of 115 000 +/- 7000 and of Stokes radius of 50-55 A. The results indicate an asymmetric shape of both delipidated monomer and dimer. Solubilization of ATPase vesicles by deoxycholate at high protein dilutions leads to almost instantaneous loss of ATPase activity. However, ATPase may be solubilized by deoxycholate in presence of phospholipid and sucrose in a temporarily active state. Inactivation appears to be accompanied by delipidation and conformational changes of the protein as evidenced by circular dichroism measurements. Sedimentation velocity examination of enzymatically active preparations of soluble ATPase in presence of phospholipid and sucrose strongly suggests that the major part of enzymatic activity is derived from a monomer with an asymmetric shape. The extent of formation of soluble oligomers by column chromatography was dependent on the exact conditions used for initial solubilization of ATPase. No evidence for differences among monomer and dimer fractions was obtained by isoelectric focusing and amino acid analysis. The results of these studies are compatible with electron-microscopic studies by other authors which suggest that the ATPase has an elongated shape with limited hydrophobic contact with the membrane lipid. A resemblance of delipidated oligomers with the form in which ATPase occurs in the membrane is conjectural at present.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D003840:Deoxycholic Acid; D004789:Enzyme Activation; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D046911:Macromolecular Substances; D003840:Deoxycholic Acid; D000251:Adenosine Triphosphatases; D002118:Calcium