Matrix Metalloproteinase 9 Pro Monomer (MMP-9, Collagenase 4, Gelatinase B)

Human MMP-9 consists of 668aa with a calculated Mrof 76,240D. Due to N- and O-linked glycosylated the Mr in SDS-PAGE is about 92kD. Within the protein sequence, the following structural domains can be distinguished: An N-terminal propeptide, which confers latency to the proenzyme, a Ca2+ and Zn2+ ion-binding catalytic domain containing an insertion of three repeats homologous to type II repeats in the gelatin-binding region of fibronectin, and a C-terminal hemopexin-like domain. Catalytic and hemopexin domains are connected by a proline-rich sequence with homology to sequences in collagens. The gelatin-binding region and the hemopexin domain confer specific macromolecular substrate binding to MMP-9. The hemopexin domain of the latent enzyme binds TIMP-1. \nActivation of latent MMP-9 can be mediated by proteases like stromelysin, cathepsin G, kallikrein and trypsin or by incubation with APMA. In the presence of APMA, the propeptide is not removed completely, however, and there occurs considerable C-terminal self-processing. The enzyme is inhibited by TIMP-1 and TIMP-2. \nMMP-9 is secreted from macrophages, polymorphonuclear leukocytes, keratinocytes and many tumor cells. It is detected in human plasma and saliva. \nMMP-9 is involved in physiological processes such as angiogenesis, wound healing, bone remodeling, migration of macrophages and leukocytes. Hydrolysis of type IV collagen and other matrix proteins in basement membranes by MMP-9 contributes to tumor cell invasion and aortic aneurysm formation.\n\nInhibitors:\nMMP-9 is inhibited by TIMPs and by chelators of divalent cations, such as EDTA or o-phenanthroline.\n\nSpecific Activity: \nThe specific activity of activated MMP-9 after trypsin activation is (same/more than) 450mU/mg, where 1U is the activity that hydrolyzes 1umol peptide (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-(3-[2, 4- dinitrophenyl]-L-2, 3-diamino-propionyl)- Ala-Arg-NH3 (Mca-Pro-Leu-Gly-Leu-Dpa- Ala-Arg) within 1 minute under the assay conditions described by Knight, et al.\n\nActivation: \nAn aliquot of 10ul MMP-9 monomer is mixed with 20ul trypsin solution (see below) and activation buffer in a total volume of 100ul. The mixture is incubated for 20 min at 37 degrees C. Thereafter trypsin is inhibited by addition of 10ul aprotinin solution. \nTrypsin Solution: 0.50mg TPCK-trypsin/ml activation buffer. The solution is stored in aliquots at -20 degrees C.\nActivation Buffer: 50mM Tris-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2. \nAprotinin Solution: 1mg aprotinin/ml activation buffer. The solution is stored at -20 degrees C.\n\nStorage and Stability:\nAliquot to avoid repeated freezing and thawing, and freeze at -70 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 6 months.

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SPECIFICATIONS

Catalog Number

M2425-40

Size

5ug

Form

Supplied as a liquid in 50mM Tris-HCl, pH 7.0, 200mM sodium chloride, 5mM CaCl2, ,1mM ZnCl2, 0.05% Brij-35, 0.05% sodium azide.

Purity

> 95% of total protein. MMP-9 monomer appears as a major band at 92kD in non-reducing SDS-PAGE.

References

1. Masui, Y., et al., Biochem. Med. 17:215-221 (1977). 2. Wilhelm, S.M., et al., J. Biol. Chem. 264:17,213-17,221 (1989). 3. Collier, I.E., et al., Genomics 9:429-434 (1991). 4. Goldberg, G.I., et al., J. Biol. Chem. 267:4583-4591 (1992). 5. Kolkenbrock, H., et al., Biol. Chem. Hoppe-Seyler 376:495-500 (1995). 6. Murphy, G. and Grabbe, T., Methods Enzymol. 248:470-484 (1995). 7. Hibbs, M.S., et al., J. Collagen Rel. Res. 4:467-477 (1984). 8. Salo, T., et al., J. Biol. Chem. 266:11,436-11,441 (1991). 9. Sato, H., et al., Oncogene 7:77-83 (1992). 10. Zucker, S., et al., Cancer Res. 53:140-146 (1993). 11. Makela, M., et al., J. Dent. Res. 73:1397-1406 (1994). 12. Shapiro, S.D., Current Opinion Cell Biol. 10:602-608 (1998). 13. Bernhard, E.J., et al., PNAS USA 91:4293-4297 (1994). 14. Thompson, R.W., J. Clin. Invest. 96:318-326 (1995). 15. Knight, C.G., et al., FEBS Lett. 296:263-266 (1992).