Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences.
Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.
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Department of Biochemistry, J.H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0581, USA.
Johnson
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D053802:Tryptases
D007527:Isoenzymes; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases