Purification, characterization and proteinase-inhibitory activity of a Clara-cell secretory protein from the pulmonary extracellular lining of rabbits.
A low-Mr Clara-cell secretory protein, CCSP, previously shown to be a major secretory product of Clara cells, was isolated from rabbit lung lavage effluents. CCSP accounted for 4.4 +/- 0.5% of the protein in the soluble phase of cell- and surfactant-free pulmonary lavage effluents (LE). Purification of this protein from LE was achieved in two steps. First, the LE was acidified with HCIO4 and, secondly, CCSP was isolated by gel-exclusion chromatography on Sephadex G-50. Purified CCSP was homogeneous by SDS/polyacrylamide-gel electrophoresis (PAGE), consisting of a single major isoform with a pI of 6.0. The Mr of CCSP was 5800 according to SDS/PAGE under reducing conditions and 12,600 under non-reducing conditions. However, by gel chromatography the Mr of the protein under non-reducing conditions was 12,400 and under reducing conditions it increased to 15,000. The discrepancy obtained by using these two techniques was attributed to anomalous electrophoretic mobilities of the protein in its reduced state. The molecule contained three half-cystine residues, but no free thiol groups, and tryptophan was not detectable. The first seven N-terminal amino acid residues were Gly-Ile-Xaa-Pro-Arg-Phe-Ala-. The third residue was not identified. CCSP showed inhibitory activity against the thiol proteinase papain (50% inhibition at 4 microM-CCSP), but only weak activities against human polymorphonuclear-leucocyte elastase, and bovine trypsin. The molecule was not digested by, and did not complex with, trypsin. CCSP was immunochemically different from surfactant apoprotein B (Mr 10,000) present in rabbit lung surfactant. This study is the first partial characterization of the major secretory protein of rabbit lung Clara cells.
Gupta
R P
RP
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Patton
S E
SE
Jetten
A M
AM
Hook
G E
GE
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000032:analysis; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D008168:Lung / Q000032:analysis*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D011817:Rabbits; D014598:Uteroglobin
D000596:Amino Acids; D011480:Protease Inhibitors; D011506:Proteins; C556389:SCGB1A1 protein, human; D014598:Uteroglobin