Purification, characterization, and localization of a novel trypsin-like protease found in the human airway.
A novel trypsin-like protease was purified to homogeneity from the sputum of patients with chronic airway diseases, by sequential chromatographic procedures. The enzyme migrated on SDS-polyacrylamide gel electrophoresis to a position corresponding to a molecular weight of 28 kDa under both reducing and non-reducing conditions, and showed an apparent molecular weight of 27 kDa by gel filtration, indicating that it exists as a monomer. It had an NH2-terminal sequence of Ile-Leu-Gly-Gly-Thr-Glu-Ala-Glu-Glu-Gly-Ser-Trp-Pro-Trp-Gln-Val-Ser-Leu- Arg-Leu, which differed from that of any known protease. Studies with model peptide substrates showed that the enzyme preferentially cleaves the COOH-terminal side of arginine residues at the P1 position of certain peptides, cleaving Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide most efficiently and having an optimum pH of 8.6 with this substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate, leupeptin, antipain, aprotinin, and soybean trypsin inhibitor, but hardly inhibited by secretory leukocyte protease inhibitor at 10 microM. An immunohistochemical study indicated that the enzyme is located in the cells of the submucosal serous glands of the bronchi and trachea. These results suggest that the enzyme is secreted from submucosal serous glands onto the mucous membrane in patients with chronic airway diseases.
Yasuoka
S
S
The Department of Nursing, School of Medical Sciences, The University of Tokushima, Japan.
Ohnishi
T
T
Kawano
S
S
Tsuchihashi
S
S
Ogawara
M
M
Masuda
K
K
Yamaoka
K
K
Takahashi
M
M
Sano
T
T
D000595:Amino Acid Sequence; D001980:Bronchi / Q000201:enzymology*; D001982:Bronchial Diseases / Q000201:enzymology*; D003374:Coumarins / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012704:Serous Membrane / Q000201:enzymology; D013183:Sputum / Q000201:enzymology; D013379:Substrate Specificity; D014132:Trachea / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry
D003374:Coumarins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C048055:tertiary-butyloxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumarin-7-amide; D005340:Fibrinogen; D012697:Serine Endopeptidases; D014357:Trypsin