Solubilization, purification, and reconstitution of alpha 2 beta 1 isozyme of Na+/K+ -ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100.
We identified alpha(2), alpha(1), and beta(1) isoforms of Na(+)/K(+)-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C(12)E(8), whereas C(12)E(8) was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase elicited higher E1Na-E2 K transition compared with that of the C(12)E(8)- and Triton X-100-purified enzyme. The rate of Na(+) efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C(12)E(8)-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase possessed more organized secondary structure compared to the C(12)E(8)- and Triton X-100-purified isozyme.
Ghosh
Biswarup
B
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, 741235, India.
Chakraborti
Tapati
T
Kar
Pulak
P
Dey
Kuntal
K
Chakraborti
Sajal
S
D000818:Animals; D002417:Cattle; D021941:Caveolae / Q000737:chemistry / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000033:anatomy & histology / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D017830:Octoxynol / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010742:Phospholipid Ethers / Q000737:chemistry; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D003902:Detergents; D007527:Isoenzymes; D008081:Liposomes; D010713:Phosphatidylcholines; D010742:Phospholipid Ethers; D017830:Octoxynol; D000254:Sodium-Potassium-Exchanging ATPase; C017251:1,2-oleoylphosphatidylcholine