Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme.
Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates.
Ohlsson
K
K
Lunds Universitet, Malmö Allmänna, Sjukhus.
Linder
C
C
Rosengren
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D005779:Immunodiffusion; D007249:Inflammation / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D010450:Endopeptidases; D012697:Serine Endopeptidases; D053146:Myeloblastin