Mercurial activation of human polymorphonuclear leucocyte procollagenase.
The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl2 was investigated by kinetic and sequence analysis of the reaction products. HgCl2 activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53-Val54 peptide bond to generate a collagenase species of Mr 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64-Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N-terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79-Met80 or Met80-Leu81 peptide bonds. Furthermore, activation in the presence of TIMP-1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53-Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N-terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl2 followed a three-step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.
Bläser
J
J
University of Bielefeld, Faculty of Chemistry, Department of Biochemistry, Federal Republic of Germany.
Knäuper
V
V
Osthues
A
A
Reinke
H
H
Tschesche
H
H
D000595:Amino Acid Sequence; D017364:Collagenases; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology*; D003012:Microbial Collagenase / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases
D004792:Enzyme Precursors; D006023:Glycoproteins; D009842:Oligopeptides; D019714:Tissue Inhibitor of Metalloproteinases; D008627:Mercuric Chloride; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase