Solubilization, purification, and reconstitution of ¦Á₂¦Â₁ isozyme of Na⁺/K⁺-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C₁₂E₈, and Trit

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• 作者：Biswarup Ghosh ; Tapati Chakraborti ; Pulak Kar ; Kuntal Dey and Sajal Chakraborti
• 关键词：Na+/K+ ; ATPase isozyme ; Caveolae ; Detergent ; Purification ; Reconstitution
• 刊名：Molecular and Cellular Biochemistry
• 出版年：2009
• 出版时间：March, 2009
• 年：2009
• 卷：323
• 期：1-2
• 页码：169-184
• 全文大小：509.1 KB

文摘

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 α₂β₁ 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 α₂β₁ 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.