Purification and characterization of a novel Kazal-type serine proteinase inhibitor of neutrophil elastase from sheep lung.
A Kazal-type elastase inhibitor was purified by trichloroacetic acid precipitation of sheep lung lavage fluid followed by chymotrypsin affinity and gel-filtration chromatography of the supernatant. Sheep lung elastase inhibitor (SLEI) is glycosylated. Laser desorption mass spectrometry indicated that SLEI has a molecular mass of 16.8-17.3 kDa. Partial protein sequence of SLEI and of a peptide derived from SLEI showed 31-52% and 51-66% homology at the N-terminus and at the inhibitory site respectively with Kazal-type double-headed proteinase inhibitors (bikazins). SLEI inhibited human leukocyte elastase and porcine pancreatic elastase but not human cathepsin G. It was inactivated by chloramine-T and reactivated when incubated with methionine sulfoxide peptide reductase and dithiothreitol, indicating the presence of a methionine at the active site. The concentration of SLEI in bronchoalveolar lavage fluid (BALF) and lung lymph was 0.28 microM (0.23-0.49); 0.24 microM (0.20-0.31) (median, (range), n = 5), respectively and was undetectable in plasma (< 0.03 microM) suggesting that SLEI is produced in the lung. The median molar ratios of SLEI to alpha1-proteinase inhibitor in BALF and lung lymph were 3.2 to 1 and 0.017 to 1, respectively. These results indicate that SLEI probably makes an important contribution to antielastase defence in epithelial lining liquid.
Mistry
R
R
Department of Medicine, Charing Cross and Westminster Medical School, London, UK. r.mistry@udcf.gla.ac.uk
Snashall
P D
PD
Totty
N
N
Briskin
S
S
Guz
A
A
Tetley
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D002335:Carnivora; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002918:Chymotrypsin; D004285:Dogs; D005589:Foxes; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D008168:Lung / Q000737:chemistry / Q000502:physiology*; D008907:Mink; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D012756:Sheep; D013552:Swine; D007507:Therapeutic Irrigation; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*
D015842:Serine Proteinase Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase