A SOLUBLE ECTO-ATPASE FROM TETRAHYMENA-THERMOPHILA - PURIFICATION ANDSIMILARITY TO THE MEMBRANE-BOUND ECTO-ATPASE OF SMOOTH-MUSCLE

For the first time, a soluble, dedicated E-type ecto-ATPase has been i dentified and purified. This fully soluble ecto-ATPase is released int o the growth media of the single-celled eukaryote, Tetrahymena, at a c onstant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for t he membrane-bound ecto-enzyme in Tetrahymena. It was purified by a com bination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determ ined to be approximately 66,000 Da by denaturing gel electrophoresis a nd approximately 69,000 Da by size exclusion chromatography of the nat ive form. The purified soluble enzyme displays the general characteris tics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence , hydrolysis of ATP and other nucleoside triphosphates (but not nucleo side diphosphates) and insensitivity to common ATPase inhibitors (vana date, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfo nate). It was further shown to be immunologically similar (by polyclon al antibodies) to both the membrane-bound ecto-ATPase of chicken gizza rd smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma m embranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fraction s but the body membrane had slightly higher specific activities. We pr opose that this ecto-ATPase of Tetrahymena may play a role in inactiva ting purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular n ucleotide scavenging. (C) 1997 Academic Press