IRREVERSIBLE INHIBITION OF HUMAN NATURAL-KILLER-CELL NATURAL CYTOTOXICITY BY MODIFICATION OF THE EXTRACELLULAR MEMBRANE BY THE ADENINE-NUCLEOTIDE ANALOG 5'-P-(FLUOROSULFONYL)BENZOYL ADENOSINE

Extracellular adenine nucleotides are inhibitors of the human natural killer cell line NK3.3 natural cytotoxicity activity. Natural cytotoxi city was inhibited approximately 26% by 1 mM ATP and 21% by 1 mM ADP. 5'-Adenylyl imidodiphosphate, a nonhydrolyzable ATP analog, inhibited natural cytotoxicity by 41% at a concentration of 1 mM and > 97 % at a concentration of 10 mM. In contrast, AMP was not inhibitory. Adenosin e was a weak inhibitor of natural cytotoxicity and may represent an al ternate regulatory pathway, Removal of the nucleotides resulted in the restoration of control levels of natural cytotoxicity activity. The a ffinity label 5'-p-(fluorosulfonyl)benzoyladenosine (5'-FSBA) is a syn thetic analog of ATP or ADP containing an electrophilic fluorosulfonyl group capable of covalently modifying proteins at adenine di- and tri phosphate nucleotide-binding sites. Natural cytotoxicity was irreversi bly inhibited by modification of the extracellular membrane of NK3.3 c ells by 5'-FSBA. This inhibition was concentration dependent with an I -50 similar to 100 mu M and complete inhibition at 1 mM. Modification of NK3.3 by 5'-FSBA did not affect the formation of effector-target ce ll conjugates; however, granule release was inhibited, This targets th e site of inhibition by 5'-FSBA modification to a pathway preceding gr anule release, Irreversible, covalent modification of surface adenine nucleotide-binding proteins by 5'-FSBA provides a probe to study the r ole of specific adenine nucleotide-binding proteins in the extracellul ar regulation of natural killer cytolytic activity by adenine nucleoti des. (C) 1995 Academic Press, Inc.