Inactivation of human lung tryptase: evidence for a re-activatable tetrameric intermediate and active monomers.
Human lung tryptase (HLT), a trypsin-like serine proteinase stored as an active enzyme in association with heparin in mast cell granules, is released into the extracellular environment when mast cells are activated. Tryptases are unusual in that they form tetramers and bind heparin. As there are no known endogenous tryptase inhibitors, loss of heparin and dissociation of the active tetrameric enzyme to inactive monomers has been proposed as the mechanism of control. Activity and intrinsic fluorescence were used to measure the stabilization of HLT by NaCl, glycerol, and heparin. At physiological salt concentrations in the absence of heparin, activity decayed rapidly (t1/2 = 1-4 min at 37 degrees C) to an intermediate that could be immediately reactivated by heparin. But protein structural changes, as measured by intrinsic fluorescence, were much slower (t1/2 = 16 min), indicating that the intermediate continued to exist as a tetramer that slowly changed to a monomer. HLT tetramers, either active or inactive, were stabilized by 2 M NaCl, 20% glycerol, and heparin. Maximum stabilization was obtained with approximately 1 mol of heparin per HLT subunit. Heparan sulfate also stabilized HLT activity and active HLT was bound to and recovered from cartilage. Subunits of the inactive intermediate appeared to be loosely associated as demonstrated by the rapid disappearance of the tetramer in gel filtration studies in 1 M NaCl (t1/2 = 1.8 min), but the tetramer was stable in lower ionic strength buffers containing heparin. Fluorescence anisotropy measurements in the absence of heparin were also consistent with a slow (t1/2 = 22 min) transition from tetramer to monomer, and native polyacrylamide gel electrophoresis provided additional evidence for a tetrameric intermediate. HLT monomers isolated by gel filtration were minimally active in the presence of heparin. These data show that heparin-free HLT rapidly converts to an "inactive", loose tetrameric intermediate that can be reactivated with heparin or slowly dissociate to less active monomers and that tryptase released from mast cells is likely to remain active in association with heparin or other extracellular components. Thus, tryptase affinity for glycosaminoglycans and substrate specificity limitations are the primary factors controlling the proteolytic functions of these enzymes.
Addington
A K
AK
Department of Biochemistry and Molecular Biology, J.H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0581, USA.
Johnson
D A
DA
D002850:Chromatography, Gel; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D005990:Glycerol / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D053802:Tryptases
D006025:Glycosaminoglycans; D012965:Sodium Chloride; D006493:Heparin; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases; D005990:Glycerol