Heinz Reinke

A 25 kDa α2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase

Besides the monomeric mammalian 95 kDa progelatinase, two additional forms, a disulfide‐bridged 220 kDa dimer and a 125 kDa form were isolated from human PMN leukocytes. The 125 kDa progelatinase was identified as a covalently linked, disulfide‐bridged heterodimer formed of the monomer with a 23 kDa protein. This 25 kDa protein was isolated from gelatinase bound to the affinity support of gelatin‐Sepharose and eluted by DTE‐containing buffer. The amino acid sequence of tryptic peptides of this protein revealed homology with an α2‐microglobulin‐related protein from rats, a protein so far unknown in humans.

Inactivation of human plasma α1‐proteinase inhibitor by human PMN leucocyte collagenase

Highly purified human polymorphonuelear leucocyte collagenase cleaved human α‐1‐proteinase inhibitor (α1‐PI) at the carboxyl site of Phe352 (P7). The inhibitor was thereby rapidly inactivated and generated a primary degradation product as shown by reverse‐phase HPLC and N‐terminal sequencing. Prolonged incubation of the modified inhibitor with polymorphonuclear leucocyte collagenase led to the generation of a secondary degradation product with additional cleavage at the carboxyl site of Pro357 (P2).

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase)

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N‐terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15‐Leu16, Glu40‐Met41, Leu52‐Leu53 and Ala74‐Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78‐Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C‐terminal cleavages. The Glu666‐Leu667, Ala506‐Glu507 and Ala398‐Leu399 bonds were hydrolysed sucessively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C‐terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin‐like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid

The tissue inhibitors of metalloproteinases TIMP‐1 and TIMP‐2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non‐covalent gelatinase/TIMP complexes. TIMP‐1 migrated as a single polypeptide with M r 28 500 on SDS‐PAGE, while the M r of TIMP‐2 was 21 000. The inhibitory activity was stable under heat and acid pH. N‐terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP‐1 and TIMP‐2. This is the first demonstration that TIMP‐1 and TIMP‐2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1‐inhibitor at the carboxyl site of Ala439 (P6). This led to a concomitant loss of C1‐inhibitor activity. An additional cleavage site at the carboxyl site of Ser441 (P4), was observed during PMN leucocyte gelatinase‐induced inactivation, and a minor fragment of the plasma C1‐inhibitor was generated.

Formation of a covalent Hg-Cys-bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine-switch mechanism

A common method for the activation of mammalian matalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three‐step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N‐terminal sequence determination proved cleavage of the Asp64‐Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine‐switch mechanism involving Cys71.

Leukodiapedesis, compartmentalisation and secretion of PMN leukocyte proteinases, and activation of PMN leukocyte procollagenase

Human polymorphonuclear leukocytes (PMNL) are the first phagocytic cells to arrive at the site of injury of the epithelium, where they regulate the defense reactions against invading microorganisms. Therefore, their accumulation characterises an inflammatory process. Some particular properties of PMNL (Fig. 1) such as phagocytising microorganisms and eliminating damaged tissue are described [1].