title: Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes.
abstract: Rat RNK-16 leukemia cells kill YAC-1, which are the cells lysed by rodent natural killer lymphocytes. We found chymotrypsin-like proteinase ('chymase') activity in the RNK-16 dense granules that also contain cytolytic activity. The chymase activity hydrolyzed the thiobenzyl peptide substrate Suc-Phe-Leu-Phe-SBzl and, in comparison to RNK-16 tryptase activity, was selectively inhibited by three different types of serine proteinase inhibitors. The selective inhibitors were the fungal aldehyde chymostatin, the chloromethylketone Z-Gly-Leu-Phe-CH2Cl, and the mechanism-based or 'suicide' inhibitor 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin. These proteinase inhibitors also blocked RNK-16 granule-mediated cytolysis. Chymostatin, a reversible inhibitor, delayed granule-mediated cytolysis, whereas the irreversible chloromethylketone and isocoumarin proteinase inhibitors completely abrogated granule-mediated cytolysis. The two irreversible inhibitors displayed biphasic inhibition of the chymase activity, indicating that at least two chymases are present in the granules. By Northern blot analysis, we found that RNK-16 mRNA hybridized strongly with a cDNA probe of CCPI, a mouse cytotoxic T lymphocyte serine proteinase gene. These data imply that chymase activity in the cytotoxic granules is important for cytolytic function and is likely to belong to a new subfamily of serine proteinases.
authors.affiliation: Cell and Molecular Biology Program, School of Medicine, University of Nevada, Reno 89557-0046.
authors.forename: S J
authors.initials: SJ
authors.lastname: Zunino
authors.forename: N J
authors.initials: NJ
authors.lastname: Allison
authors.forename: C M
authors.initials: CM
authors.lastname: Kam
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
authors.forename: D
authors.initials: D
authors.lastname: Hudig
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D053818:Chymases; D003594:Cytoplasmic Granules / Q000201:enzymology; D005796:Genes; D006461:Hemolysis; D007942:Leukemia, Experimental / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053818:Chymases