THE TRANSMEMBRANE DOMAINS OF ECTOAPYRASE (CD39) AFFECT ITS ENZYMATIC-ACTIVITY AND QUATERNARY STRUCTURE

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 membr ane protein solubilization. In contrast, the enzymatic activities of s oluble E-type ATPases, including potato tuber (Solanum tuberosum) apyr ase and parasite ecto-ATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce th e activity of the enzyme promote dissociation of the tetramer to monom ers. 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 a nd its activity is not affected by detergents. Mutants of the ectoapyr ase with only the NH2- or the COOH-terminal transmembrane domain are m embrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than tha t 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.