Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase.
Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.
King
C H
CH
Department of Medicine, Case Western Reserve University, Cleveland, OH 44106.
Hull
A
A
Kleinhenz
P J
PJ
Phillips
N F
NF
Marino
J A
JA
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010450:Endopeptidases