PURIFICATION AND CHARACTERIZATION OF A HUMAN BRADYKININ BINDING-PROTEIN FROM INFLAMMATORY CELLS

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmaco logical and inflammatory actions which are exerted through cell surfac e 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 lymph ocytes. Radioreceptor crosslinking experiments using bifunctional cros slinkers and radiolabelled BK identified a 14 kDa protein in these cel l types both on the cell surface, in glycerol purified plasma membrane s and in detergent solubilized cell extracts. Purification by BK affin ity 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 demonst rated three major proteins at 190, 50 and 14 kDa when eluted with eith er excess BK or mild acid. Neutrophil fractions from detergent solubil ized 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. Radiorecep tor dot assays of the purified neutrophil eluates containing the 14 kD a protein revealed specific binding to [I-125]-BK with a 160 fold exce ss signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific b inding protein expressed on the surface of inflammatory cells. (C) 199 8 Published by Elsevier Science Ltd. All rights reserved.