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 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.
Wang
T F
TF
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Ou
Y
Y
Guidotti
G
G
D000227:Adenine Nucleotides / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010271:Parasites; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology; D013379:Substrate Specificity; D014162:Transfection
D000227:Adenine Nucleotides; D015703:Antigens, CD; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen