A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme in Tetrahymena. It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ecto-ATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase of Tetrahymena may play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
Smith
T M
TM
Department of Biological Sciences, State University of New York at Buffalo, New York 14260, USA.
Kirley
T L
TL
Hennessey
T M
TM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002923:Cilia / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism; D012995:Solubility; D016808:Tetrahymena thermophila / Q000201:enzymology*
D009711:Nucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D008274:Magnesium; D002118:Calcium