A Highly Sensitive Immunoenzymometric Assay for the Determination of Angiogenin

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FORMATION OF A COVALENT HG-CYS-BOND DURING MERCURIAL ACTIVATION OF PMNL PROCOLLAGENASE GIVES EVIDENCE OF A CYSTEINE-SWITCH MECHANISM

BLASER J, TRIEBEL S, REINKE H, Tschesche H. FORMATION OF A COVALENT HG-CYS-BOND DURING MERCURIAL ACTIVATION OF PMNL PROCOLLAGENASE GIVES EVIDENCE OF A CYSTEINE-SWITCH MECHANISM. FEBS LETTERS. 1992;313(1):59-61.A common method for the activation of mammalian metalloproteinases 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

A 25 KDA ALPHA-2-MICROGLOBULIN-RELATED PROTEIN IS A COMPONENT OF THE 125-KDA FORM OF HUMAN GELATINASE

TRIEBEL S, BLASER J, REINKE H, Tschesche H. A 25 KDA ALPHA-2-MICROGLOBULIN-RELATED PROTEIN IS A COMPONENT OF THE 125-KDA FORM OF HUMAN GELATINASE. FEBS LETTERS. 1992;314(3):386-388.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 hetrodimer formed of the monomer with a 25 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 alpha2-microglobulin-related protein from rats, a protein so far unknown in humans

MERCURIAL ACTIVATION OF HUMAN-PMN LEUKOCYTE TYPE-IV PROCOLLAGENASE (GELATINASE)

TRIEBEL S, BLASER J, REINKE H, KNAUPER V, Tschesche H. MERCURIAL ACTIVATION OF HUMAN-PMN LEUKOCYTE TYPE-IV PROCOLLAGENASE (GELATINASE). FEBS LETTERS. 1992;298(2-3):280-284.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