Solubilization, purification, and reconstitution of α₂ β₁ isozyme of Na⁺/K⁺-ATPase from caveolae of pulmonary smooth muscle plasma membrane: Comparative studies with DHPC, C₁₂E₈, and Triton X-100

We identified α₂, α₁, and β₁ isoforms of Na⁺/K⁺-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α₂ β₁ 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 (C₁₂E₈), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C₁₂E₈, whereas C₁₂E₈ was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α₂ β₁ isozyme of Na⁺/K⁺-ATPase elicited higher E1Na-E2 K transition compared with that of the C₁₂E₈- and Triton X-100-purified enzyme. The rate of Na⁺ efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C₁₂E₈-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α₂β₁ isozyme of Na⁺/K⁺-ATPase possessed more organized secondary structure compared to the C₁₂E₈- and Triton X-100-purified isozyme.