Spontaneous inactivation of human lung tryptase as probed by size-exclusion chromatography and chemical cross-linking: dissociation of active tetrameric enzyme into inactive monomers is the primary event of the entire process.
A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of apparent equilibrium between all three forms (active tetramer, inactive monomer, inactive tetramer) was reached. When tryptase was extensively cross-linked with several heterobifunctional photoactivatable reagents, this modified enzyme exhibited a long-term stability in low-ionic-strength buffer and at elevated temperature, unlike that of the native enzyme. Its is suggested that cross-linking prevents the spontaneous inactivation and dissociation of tryptase by 'freezing' the normal association state of the enzyme and supports the hypothesis that the dissociation of native tetrameric tryptase into inactive monomer is the primary event for the entire process of spontaneous inactivation of this enzyme.
Kozik
A
A
Institute of Molecular Biology, Jagiellonian University, Kraków 31-120, Poland.
Potempa
J
J
Travis
J
J
D001704:Biopolymers / Q000378:metabolism; D002850:Chromatography, Gel; D053818:Chymases; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D009994:Osmolar Concentration; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D053802:Tryptases
D001704:Biopolymers; D003432:Cross-Linking Reagents; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases