Purification of tryptase from a human mast cell line.
The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.
Butterfield
J H
JH
Department of Internal Medicine, Mayo Medical School, Rochester, Minnesota.
Weiler
D A
DA
Hunt
L W
LW
Wynn
S R
SR
Roche
P C
PC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007946:Leukemia, Mast-Cell / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008415:Mastocytosis / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D008627:Mercuric Chloride / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000032:analysis / Q000302:isolation & purification*; D012867:Skin / Q000166:cytology
D000590:Amino Acid Chloromethyl Ketones; D000596:Amino Acids; D003619:Dansyl Compounds; D008627:Mercuric Chloride; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D005990:Glycerol