Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine.
The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.
Shivanna
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Veterans Affairs Medical Center, 4801 Linwood Blvd, Kansas City, MO 64128, USA.
Rowe
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ES
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D019826:Cholic Acid; D002793:Cholic Acids; D002942:Circular Dichroism; D003840:Deoxycholic Acid; D003902:Detergents; D005227:Fatty Acids / Q000032:analysis; D005260:Female; D005453:Fluorescence; D008081:Liposomes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology; D010713:Phosphatidylcholines; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012542:Scattering, Radiation; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D005227:Fatty Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D000077423:Polidocanol; C038470:1,2-heptanoylphosphatidylcholine; D011092:Polyethylene Glycols; D000252:Calcium-Transporting ATPases; C017251:1,2-oleoylphosphatidylcholine; D019826:Cholic Acid; D002118:Calcium