Besides the mnnomcric mammalian 95 kDa progelatinase, two additional forms, ~1 disulfide-bridged 220 kDa dinxr and a 125 kDn form were isolated from human PMN Icukocytes. The I25 kDa progelatinase was identified as a covalcntly linked, disul!!de-bridged hetcrodimer formed of the monomer with a 2s kDa protein. This 25 kDa protein was isolntcd from pclatinase bound to the atIinity support of gelatin-Scpharosc and eluted by DTE-containing bulli3r. The amino acid sequence of tryptic peptides of this protein rcvenled homology with an c+-microglobulin-related protein from rats, a protein so far unknown in humans.

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl 2 -and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.

A polyclonal antibody to human recombinant angiogenin was prepared in rabbits using a Pam 3 CysSerGly conjugate. The antibody was then used to develop the first highly sensitive enzyme-labelled immunometric assay for this vascularisation inducing and tumour associated protein. The assay was suitable for quantification of angiogenin in body fluids between 2.5 and 0.05 g/l. The mean intra-assay imprecision was 6.0% and the inter-assay imprecision 7.9%. Angiogenin in human plasma was found to lie in the range of 0.38 to 0.11 mg/1 with a mean of 0.25 + 0.07 mg/1.

No abstract

TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)