title: Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form.
abstract: Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.
authors.affiliation: Department of Biophysical Chemistry, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
authors.forename: A I
authors.initials: AI
authors.lastname: Marcy
authors.forename: L L
authors.initials: LL
authors.lastname: Eiberger
authors.forename: R
authors.initials: R
authors.lastname: Harrison
authors.forename: H K
authors.initials: HK
authors.lastname: Chan
authors.forename: N I
authors.initials: NI
authors.lastname: Hutchinson
authors.forename: W K
authors.initials: WK
authors.lastname: Hagmann
authors.forename: P M
authors.initials: PM
authors.lastname: Cameron
authors.forename: D A
authors.initials: DA
authors.lastname: Boulton
authors.forename: J D
authors.initials: JD
authors.lastname: Hermes
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003012:Microbial Collagenase / Q000235:genetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004792:Enzyme Precursors; D006877:Hydroxamic Acids; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D003012:Microbial Collagenase