Identification and characterization of multiple forms of tryptase from human mast cells.
Mast cell tryptase purified from human adult skin (AS), adult lung (AL) and newborn foreskin (NS) with a monoclonal antitryptase B2 immunoaffinity Sepharose column was further fractionated by HPLC using a Mono-S cation exchange column at pH 6.5. Tryptases exhibited two clearly separated major fractions, both of which also revealed at least two overlapping peaks. Native tryptase molecules from skin consisted of two diffuse protein bands in SDS-PAGE at about 31 and 35 kDa, whereas those from lung usually exhibited a predominant diffuse band at about 29 kDa. The forms of tryptases separated by Mono-S HPLC gave a different banding pattern in SDS-PAGE. Tryptase from NS exhibited chromatographic peaks that each showed Mr values approximately 1-3 kDa higher than those of tryptase from AS. By gel filtration, the Mr values for native major fractions of tryptases derived from AS and AL were 178 kDa and 141 kDa, respectively. After carbohydrate removal by glycanase, the observed differences in Mr values in SDS-PAGE reduced to two similar sharp bands of Mr approximately 28 kDa and 30 kDa for all tryptase preparations. AS and AL tryptases and their subfractions exhibited similar enzyme kinetic values and similar immunoreactivities in a tryptase immunoassay. Inactivation rates at physiologic ionic strength were similar for both AL and AS tryptases. The results show the enzymatic and antigenic similarity between lung and skin tryptases, and suggest that tryptase is stored mainly as beta-tryptase in human mast cells. Tryptase immunoassay measures similarly both lung and skin tryptases and, thus, this assay is suitable for detection of mast cell activation, in contrast to assays for other proteinases of mast cells, e.g. chymase, cathepsin G and carboxypeptidase, that are present in MC(TC) cells mainly in skin only.
Harvima
R J
RJ
Department of Internal Medicine, Medical College of Virginia, Richmond, USA.
Harvima
I T
IT
Dull
D
D
Dunder
U K
UK
Schwartz
L B
LB
D000328:Adult; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007231:Infant, Newborn; D007527:Isoenzymes / Q000032:analysis*; D008168:Lung / Q000201:enzymology; D008297:Male; D008407:Mast Cells / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000032:analysis*; D012867:Skin / Q000201:enzymology; D013997:Time Factors; D053802:Tryptases
D007527:Isoenzymes; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases