Effect of histamine and divalent cations on the activity and stability of tryptase from human mast cells.
Tryptase from human mast cells is stabilized by negatively charged macromolecules such as heparin and is not affected by the protein inhibitors of serine proteinases normally present in human extracellular fluids. The current study demonstrated inhibition of tryptase-catalyzed cleavage of tosyl-Gly-Pro-Lys-p-nitroanilide by histamine and calcium, and destablization only by calcium. Calcium-mediated inhibition was competitive with a Ki of 30 mM. Cooperation of calcium with other extracellular cations or concentrations of calcium possible within cells or granules may permit calcium-mediated inhibition to occur in vivo. In contrast, only 5 mM calcium is needed to cause an irreversible 50% loss of tryptase activity after 60 min at room temperature. Histamine and N-methyl histamine concentrations of 2 mM to 10 mM inhibited tryptase activity by a different mechanism than calcium, resulting in sigmoid rather than hyperbolic kinetics. Whether this reflects cooperative binding of histamine to tryptase or conformational alterations of tryptase is not known. These concentrations of histamine are most relevant to those in mast cell secretory granules estimated at 100 mM, where tryptase is stored fully active and where histamine may play a role in attenuating tryptase activity.
Alter
S C
SC
Department of Medicine, Medical College of Virginia, Richmond 23298.
Schwartz
L B
LB
D002413:Cations, Divalent / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008407:Mast Cells / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors
D002413:Cations, Divalent; D008670:Metals; D011480:Protease Inhibitors; D006632:Histamine; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase