Purification and characterization of a human bradykinin binding protein from inflammatory cells.
Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.
Rajasekariah
P
P
Department of Immunology, Repatriation General Hospital Concord, University of Sydney, NSW, Australia.
Warlow
R S
RS
Campbell
M E
ME
Ozsarac
N
N
Dao
P L
PL
Swanton
M K
MK
Walls
R S
RS
D001920:Bradykinin / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D018836:Inflammation Mediators / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism; D011869:Radioligand Assay; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D018836:Inflammation Mediators; D008565:Membrane Proteins; D018002:Receptors, Bradykinin; D001920:Bradykinin