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.

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain.

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices. We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

Isolation of a clone partially encoding hill kangaroo X-linked hypoxanthine phosphoribosyltransferase: sex differences in methylation in the body of the gene.

An X-linked clone encoding exons 4-9 of the hypoxanthine phosphoribosyltransferase (HPRT) gene was isolated from a kangaroo (Macropus robustus: Marsupialia) lambda EMBL4 genomic library. Sequence similarity between the kangaroo and eutherian HPRT coding sequences was high; however, intron sizes varied significantly between the kangaroo and other eutherian species. HpaII and HhaI sites in the body of the gene were generally hypermethylated in vivo on the active, relative to the inactive X, with sites within intron 3 showing essentially complete correspondence of activity with methylation and inactivity with unmethylation. At approximately 5 kb downstream from the gene, a switch to unmethylation of active X-linked sites occurred. This switch occurred within a cluster of HpaII and HhaI sites that may represent a CG island associated with a subsequent gene.