title: Human matrix metalloproteinase-9: activation by limited trypsin treatment and generation of monoclonal antibodies specific for the activated form.
abstract: For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and activated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise removal of the propeptide domain and also caused cleavage within the C-terminal domain. Monoclonal antibodies specific for the activated form of human MMP-9 were raised by using a peptide corresponding to the N-terminus of the activated enzyme as immunogen. The antibodies do not recognise the MMP-9 proenzyme or the active or proenzyme forms of matrix metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP-9 in formalin-fixed, wax-embedded sections from a series of oesophageal cancer cases previously shown to contain MMP-9. All of the tumours contained activated MMP-9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin-fixed, wax-embedded sections, they should prove useful for the detection of activated MMP-9 in various disease processes.
authors.affiliation: Department of Molecular and Cell Biology, University of Aberdeen, UK.
authors.forename: M E
authors.initials: ME
authors.lastname: Duncan
authors.forename: J P
authors.initials: JP
authors.lastname: Richardson
authors.forename: G I
authors.initials: GI
authors.lastname: Murray
authors.forename: W T
authors.initials: WT
authors.lastname: Melvin
authors.forename: J E
authors.initials: JE
authors.lastname: Fothergill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D017364:Collagenases / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D004938:Esophageal Neoplasms / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014357:Trypsin / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D014357:Trypsin; D017364:Collagenases; D020780:Matrix Metalloproteinase 9