Ghosh Biswarup, Chakraborti Tapati, Kar Pulak, Dey Kuntal, Chakraborti Sajal
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, 741235, India.
Mol Cell Biochem. 2009 Mar;323(1-2):169-84. doi: 10.1007/s11010-008-9977-0. Epub 2008 Dec 20.
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.
我们在牛肺平滑肌质膜的小窝囊泡中鉴定出了Na(+)/K(+)-ATP酶的α(2)、α(1)和β(1)亚型。在使用去污剂1,2-庚酰基-sn-磷脂酰胆碱（DHPC）、聚氧乙烯8-月桂基醚（C(12)E(8)）和 Triton X-100进行溶解和纯化，并与磷脂二油酰磷脂酰胆碱（DOPC）重构的过程中，研究了小窝囊泡中Na(+)/K(+)-ATP酶α(2)β(1)同工酶的生化和生物物理特性。结果表明，在活性酶产量和比活性方面，DHPC优于C(12)E(8)，而C(12)E(8)优于Triton X-100。与用C(12)E(8)和Triton X-100纯化的酶相比，用DHPC纯化的Na(+)/K(+)-ATP酶α(2)β(1)同工酶的荧光研究引发了更高的E1Na-E2K转变。与用C(12)E(8)-DOPC和Triton X100-DOPC重构的酶相比，用DHPC-DOPC重构的同工酶中Na(+)外流速率更高。圆二色性分析表明，与用C(12)E(8)和Triton X-100纯化的同工酶相比，用DHPC纯化的Na(+)/K(+)-ATP酶α(2)β(1)同工酶具有更有序的二级结构。
