Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin.
Cytolytic granules purified from natural killer lymphocytes (NK) contain a pore-forming protein (perforin) and a number of serine proteases. When these proteases are inhibited by serine protease-specific isocoumarin reagents the serine proteases are inactivated and the cytolytic activity of the granules is decreased. Paradoxically, it has been found that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) frequently cannot block killing even though it inhibits many of the serine proteases. At the same time it has been reported that "purified" perforin alone can lyze cells. To address these inconsistencies we first compared the ability of PMSF and four new sulfonyl fluoride serine protease inhibitors to inhibit proteases and cell lysis. We determined the effects on lysis and the second order inhibition rate constants for five granule protease activities: ly-tryptase, ly-chymase, Met-ase (methionine cleaving), Ser-ase (serine cleaving) and Asp-ase (aspartic acid cleaving). One compound, 2-(Z-NH(CH2)2CONH)C6SO2F, was a potent inhibitor of Met-ase activity (k(obsd)/[I] = 162 M-1 s-1), ly-chymase activity (k(obsd)/[I] = 147 M-1 s-1), and granule-mediated as well as perforin-mediated lysis. PMSF was a poor inhibitor of granule proteases (k(obsd)/[I]'s less than 7 M-1 s-1 for four activities and no inhibition of Ser-ase); the lack of reactivity is consistent with the failure of PMSF to block granule lytic activity. We also prepared enriched perforin by anion exchange chromatography and showed that a ly-chymase and a Met-ase associated with perforin. By inhibiting these proteases we also inhibited lytic activity.
Ewoldt
G R
GR
School of Medicine, University of Nevada, Reno 89557.
Winkler
U
U
Powers
J C
JC
Hudig
D
D
D000818:Animals; D003594:Cytoplasmic Granules / Q000201:enzymology*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000201:enzymology*; D007700:Kinetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology*; D054353:Perforin; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D052899:Pore Forming Cytotoxic Proteins; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D015842:Serine Proteinase Inhibitors; D054353:Perforin; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases