Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP.
Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.
Filippini
A
A
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Taffs
R E
RE
Agui
T
T
Sitkovsky
M V
MV
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology*; D013861:Thiocyanates / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D013861:Thiocyanates; C022571:adenosine 5'-O-(3-thiotriphosphate); C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D000241:Adenosine; D002118:Calcium