Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome c oxidase
Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome c oxidase
复制标题
DOI：
10.1021/bi016080g
复制
发表时间：
2002-04-02
期刊：
影响因子：
2.9
通讯作者：
Robinson, NC
中科院分区：
文献类型：
--
作者：
Musatov, A;Robinson, NC
牛心脏细胞色素 C 氧化酶 (CcO) 通过非离子去污剂或磷脂溶解，在添加胆汁盐（例如胆酸钠、脱氧胆酸钠或 CHAPS）后完全二聚化。无论十二烷基麦芽糖苷、癸基麦芽糖苷或 Triton X-100 是主要增溶去垢剂，还是酶分散在磷脂酰胆碱、磷脂酰乙醇胺或其混合物中，都会发生胆汁盐诱导的二聚化。在每种情况下，在结合去垢剂和/或磷脂校正后，可以通过沉降速度和沉降平衡来验证完全的 CcO 二聚化。胆汁盐的相对浓度对于均质二聚 CcO 的生产至关重要。例如，用 2 mM 去垢剂溶解酶需要等摩尔浓度的胆酸钠。同样，分散在 20 mM 磷脂中的酶需要 50 mM 胆酸钠，该浓度通常用于将 CcO 重构为小单层囊泡。胆汁盐的作用不仅仅是稳定二聚 CcO 和防止去污剂诱导的解离成单体。它们能够完全逆转洗涤剂引起的单体化，并导致完全单体的 CcO 重新结合。如此生成的二聚 CcO 并不比原始复合物更稳定，并且如果去除胆汁盐，则很容易解离成单体。二聚化过程取决于亚基的完整补充；例如，如果去除亚基VIa和VIb，则所得单体CcO在添加胆酸钠时将不会重新结合。这些结果支持了四个重要的结论：（1）二聚CcO解离成单体是可逆的； (2)在溶液条件下可以产生稳定的二聚体； (3)即使在较高pH和较低酶浓度下二聚体也能稳定； (4)二聚化需要亚基VIa和VIb。
Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidyleholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.