Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity.
We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000-65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10-12 micrograms kinin/hr/mg but released only 2 micrograms kinin/hr/mg at pH 7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.
Proud
D
D
Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21239.
Siekierski
E S
ES
Bailey
G S
GS
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007073:Immunoglobulin E / Q000502:physiology; D007610:Kallikreins / Q000302:isolation & purification / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D007073:Immunoglobulin E; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D007610:Kallikreins