The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence that the proteinase is serine dependent and has aromatic amino acid specificity of cleavage.
We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.
D000596:Amino Acids / Q000378:metabolism*; D001667:Binding, Competitive; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000276:immunology; D009842:Oligopeptides / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D000514:alpha 1-Antichymotrypsin
D000596:Amino Acids; D009842:Oligopeptides; D011480:Protease Inhibitors; D000514:alpha 1-Antichymotrypsin; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; C002101:chymostatin; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D004121:Dimethyl Sulfoxide