The activation of human type IV collagenase proenzyme. Sequence identification of the major conversion product following organomercurial activation.
Type IV collagenase is a metalloproteinase which cleaves type IV collagen in a pepsin-resistant domain. Organomercurial activation of the latent 70-kDa type IV collagenase (type IV procollagenase) results in the autocatalytic removal of an amino-terminal domain resulting in the conversion to a 62-kDa activated form of the enzyme. Synthetic peptides corresponding to domains from the amino terminus (residues 1-17) and an internal domain near the carboxyl terminus (residues 472-490) were used as antigens to generate affinity-purified polyclonal antibodies which recognized their respective domains on the native type IV procollagenase. Western immunoblotting studies of the time course of the organomercurial activation process demonstrate a direct loss of the amino-terminal domain during the conversion to the lower molecular weight form. The amino-terminal sequence of the purified type IV procollagenase before and after activation reveals cleavage at a single locus with removal of residues 1-80, generating a new amino terminus YNFFPRKPKWDKNQ. This results in the removal of three distal cysteine residues located at positions 31, 36, and 73. The type IV collagenase site of autocatalytic cleavage corresponds exactly to the homologous sites of type I collagenase and stromelysin cleavage during their respective organomercurial activation. This site is adjacent to the carboxyl end of a highly conserved region consisting of the sequence PRCGVPDV, which contains an unpaired cysteine residue.
Stetler-Stevenson
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WG
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Krutzsch
H C
HC
Wacher
M P
MP
Margulies
I M
IM
Liotta
L A
LA
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008545:Melanoma / Q000201:enzymology; D003012:Microbial Collagenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009941:Organomercury Compounds / Q000494:pharmacology*; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
D004792:Enzyme Precursors; D009941:Organomercury Compounds; C010024:4-aminophenylmercuriacetate; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase; D010662:Phenylmercuric Acetate