Wang T F, Ou Y, Guidotti G
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Biol Chem. 1998 Sep 18;273(38):24814-21. doi: 10.1074/jbc.273.38.24814.
Mammalian ectoapyrase (CD39) is an integral membrane protein with two transmembrane domains and a large extracellular region. The enzymatic activity of ectoapyrase is inhibited by most detergents used for membrane protein solubilization. In contrast, the enzymatic activities of soluble E-type ATPases, including potato tuber (Solanum tuberosum) apyrase and parasite ecto-ATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce the activity of the enzyme promote dissociation of the tetramer to monomers. We expressed a secreted form of the ectoapyrase in COS-7 cells by fusing the signal peptide of murine CD4 with the extracellular domain of the ectoapyrase. The soluble ectoapyrase is catalytically active and its activity is not affected by detergents. Mutants of the ectoapyrase with only the NH2- or the COOH-terminal transmembrane domain are membrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than that of the native enzyme. These results suggest that the proper contacts between the transmembrane domains of the monomers in the tetramer are necessary for full enzymatic activity.
哺乳动物胞外ATP双磷酸酶（CD39）是一种整合膜蛋白，有两个跨膜结构域和一个大的细胞外区域。胞外ATP双磷酸酶的酶活性会受到大多数用于溶解膜蛋白的去污剂的抑制。相比之下，可溶性E型ATP酶的酶活性，包括马铃薯块茎（茄属）ATP双磷酸酶和寄生虫胞外ATP酶，不受去污剂影响。在此我们表明，胞外ATP双磷酸酶是一种四聚体，降低该酶活性的去污剂会促使四聚体解离为单体。我们通过将小鼠CD4的信号肽与胞外ATP双磷酸酶的细胞外结构域融合，在COS-7细胞中表达了一种分泌形式的胞外ATP双磷酸酶。可溶性胞外ATP双磷酸酶具有催化活性，其活性不受去污剂影响。仅具有NH2 - 或COOH - 末端跨膜结构域的胞外ATP双磷酸酶突变体与膜结合，其活性不再受去污剂影响。所有突变蛋白的酶活性均低于天然酶。这些结果表明，四聚体中单体的跨膜结构域之间的适当接触对于充分的酶活性是必要的。
