The precursor of
matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa
progelatinase/type IV
procollagenase', was purified from the
conditioned medium of U937 monocytic leukaemia and HT1080
fibrosarcoma cell lines stimulated with
phorbol 12-myristate 13-acetate.
ProMMP-9 in these
culture media is non-covalently complexed with the 29 kDa
tissue inhibitor of metalloproteinases (TIMP), but free
proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of
proMMP-9 with
4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms.
Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to
alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with
4-aminophenylmercuric acetate, but no
enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight
endopeptidases (
trypsin,
chymotrypsin,
plasmin,
plasma kallikrein,
thrombin,
cathepsin G,
neutrophil elastase and
thermolysin) were tested for their ability to activate
proMMP-9. Of them,
trypsin was the most effective activator of
proMMP-9. Only partial activation (10-30%) was observed with
plasmin,
cathepsin G and
chymotrypsin. The active forms generated by
trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to
alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP,
proMMP-9 was also converted into the same molecular-mass species by
trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested
gelatin,
type-V collagen, reduced carboxymethylated
transferrin and, to a lesser extent,
type-IV collagen and
laminin A chain. The specific activity against
gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of
gelatin degraded/min at 37 degrees C) by titration with
alpha 2-macroglobulin. Comparative studies on digestion of
gelatin and
collagen types IV and V by MMP-9 and MMP-2 indicated that both
enzymes degrade these substrates into similar fragments. However, the susceptibilities of
laminin,
fibronectin and reduced carboxymethylated
transferrin to these two
MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related
proteinases.