title: Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC.
abstract: The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.
authors.affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
authors.forename: Amritlal
authors.initials: A
authors.lastname: Mandal
authors.forename: Sudip
authors.initials: S
authors.lastname: Das
authors.forename: Tapati
authors.initials: T
authors.lastname: Chakraborti
authors.forename: Pulak
authors.initials: P
authors.lastname: Kar
authors.forename: Biswarup
authors.initials: B
authors.lastname: Ghosh
authors.forename: Sajal
authors.initials: S
authors.lastname: Chakraborti
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D003902:Detergents / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D008081:Liposomes; D008722:Methods; D008861:Microsomes / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D017830:Octoxynol / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000494:pharmacology*; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011651:Pulmonary Artery / Q000166:cytology; D012995:Solubility
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D003902:Detergents; D008081:Liposomes; D010742:Phospholipid Ethers; D000077423:Polidocanol; D011092:Polyethylene Glycols; D017830:Octoxynol; D000252:Calcium-Transporting ATPases