Human skin tryptase: kinetic characterization of its spontaneous inactivation.
The spontaneous loss of human tryptase hydrolytic activity was investigated. Time course studies monitoring the loss in catalytic activity were biphasic and correlated with a reduction in the concentration of catalytic sites. There was an initial rapid phase leading to greater than 85% loss in activity. The remaining activity gradually decayed toward completion over a 40-h period. The initial phase could be described as a first-order process with a t1/2 of approximately 6.0 min in 0.2 M NaCl (pH 6.8, 30 degrees C). The rate constant for this phase showed little, if any, sensitivity to changes in enzyme concentration, consistent with a first-order process, and analysis of the reaction as a function of temperature was consistent with a single rate-determining step. The rate of this process, however, showed marked sensitivity to changes in NaCl concentration and pH. Increasing the NaCl concentration as well as decreasing the pH below the pI (pH 6.3) reduced the rate of activity loss, whereas increasing the pH above pH 8.0 markedly increased the rate of activity loss. The effect of NaCl concentration and pH on the rate of activity loss suggests that the rate-limiting step governing the fast phase of the reaction involves electrostatic interactions. The presence of a fast and a slow phase in the decay process may suggest heterogeneity in the sample or the rapid formation of an inactive, but reversible, intermediate. A reversible intermediate was demonstrated when "inactivated tryptase" was incubated in the presence of heparin, and an increase in tryptase catalytic activity was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Schechter
N M
NM
Department of Dermatology, University of Pennsylvania, Philadelphia 19104.
Eng
G Y
GY
McCaslin
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002417:Cattle; D053818:Chymases; D004220:Disulfides / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D009994:Osmolar Concentration; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity; D053802:Tryptases
D004220:Disulfides; D009842:Oligopeptides; D010450:Endopeptidases; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases