ALEXIS Biochemicals: MMP-1 Proenzyme (human)

ALX-200-418

Revised 23-Jul-07

MMP-1 Proenzyme (human)
SYNONYMS	Collagenase (Type I) (human) Matrix Metalloproteinase 1 (human) Interstitial Collagenase (human)
PRODUCT LINE	Cytoskeleton
PRODUCT CATEGORY	MMPs

Ordering Information

Product Numbers:	Format:	Size:	Unit Price:	Quantity:	Add To Cart
ALX-200-418-C005		5 µg	182.00 USD

Product Specification

MW:	~56kDa.
EC:	3.4.24.7
SOURCE/HOST:	Isolated from human rheumatoid synovial fibroblasts. Requires activation.
CONCENTRATION:	100µg/ml (Pierce-BCA).
PURITY:	≥90% (SDS-PAGE, Western blot)
PURITY DETAIL:	No other MMP contaminants are detectable.
FORMULATION:	Liquid. In 50mM TRIS-HCl, pH 7.0, 300mM NaCl, 5mM CaCl₂, 1µM ZnCl₂, 0.05% BRIJ35 and 0.05% sodium azide.
SPECIFIC ACTIVITY:	≥100mU/mg protein (Y. Masui, et al.; Biochem. Med. 17, 215 (1977)). One unit is defined as the amount of enzyme that hydrolyzes 1µmol Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH per min. at 37°C, pH 7.0. APMA activation 1 hour at 37°C; enzyme dilution for assay 1:10/5-10µl for enzyme activity assay - incubation time 30min.
SHIPPING:	SHIPPED ON DRY ICE
LONG TERM STORAGE:	-80°C
HANDLING:	Avoid freeze/thaw cycles.

Product Specific Literature References

Synthetic substrates for vertebrate collagenase: Y. Masui, et al.; Biochem. Med. 17, 215 (1977) Abstract
Secreted forms of human neutrophil collagenase: K.A. Hasty, et al.; J. Biol. Chem. 261, 5645 (1986) Abstract; Full Text
Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis: S.M. Wilhelm, et al.; PNAS 83, 3756 (1986) Abstract
Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate: H. Nagase, et al.; Biochemistry 29, 5783 (1990) Abstract
Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin): K. Suzuki, et al.; Biochemistry 29, 10261 (1990) Abstract
Stepwise activation mechanisms of the precursors of matrix metalloproteinases 1 (tissue collagenase) and 3 (stromelysin): H. Nagase, et al.; Biomed. Biochim. Acta 50, 749 (1991) Abstract
A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett. 296, 263 (1992) Abstract
The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix: B.K. Pilcher, et al.; J. Cell Biol. 137, 1445 (1997) Abstract
The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1): R. Vallon, et al.; Eur. J. Biochem. 244, 81 (1997) Abstract

General Information

MMP-1, the classical matrix metalloproteinase, is expressed by a large number of cell types. As an example, in basal keratinocytes migrating across the dermal matrix the enzyme is invariably expressed and cleaves fibrilliar collagen type I. It was shown that the interaction of the α₂β₁ integrin with dermal collagen mediates induction of MMP-1 in keratinocytes at the onset of healing and that the activity of MMP-1 is needed to initiate cell movement. The cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization (Pilcher et al. 1997).
The MMP-1 zymogens of ~56/52kDa can be activated by a stepwise mechanism through which sequential processing events occur in the propeptide region. Both proenzymes can be activated by limited digestion with trypsin or by treatment with APMA generating their respective active enzyme forms of ~46/42kDa (Wilhelm et al. 1986).

BACKGROUND/TECHNICAL INFORMATION

Activity:
We recommend to dilute this enzyme preparation at least 1:10 and to take 5µl or less for the determination of activity. Specific activity can be assayed with the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) (Masui et al.). Substrate concentration should be 0.5mg/ml in buffer 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl₂, 1µM ZnCl₂, 0.05% BRIJ35, 0.05% sodium azide, containing 0.05mg/ml albumin. One unit MMP catalyzes the hydrolysis of 1µmol Dnp-peptide/min at 37°C and pH 7.0.
We recommend to employ the fluorogenic substrate (7-Methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-N-β-Dnp-L-α,β-diaminopropionyl-Ala-Arg-NH₂) (Knight et al. 1992). The hydrolysis of the Gly-Leu bond separates the highly fluorescent (7-Methoxycoumarin-4-yl)acetyl group from the 2,4-dinitrophenyl resulting in an increase of fluorogenic intensity. The substrate should be kept as a 9.15mM stock solution in DMSO (10mg/ml). In the assay the substrate concentration should be ~25mM. The assay can be performed in a 96-well microtiter plate (200µl per well) suitable for fluorogenic measurements (excitation wavelength of 328nm; emission wavelength of 393nm).

Activation:
Do not dilute enzyme for activation! Activation is required by trypsin (2µl trypsin; 1mg/ml) for 10-20 min. at 37°C and stopped by the addition of 10µl trypsin-inhibitor (2mg/ml) or aprotinin or TLCK. Activation can also be done by 2mM (final concentration) APMA for 60 min. at 37°C.

Inhibitors:
Only the activated and not the latent forms of wild-type MMP-1 protein is able to form a complex with TIMP-1. Quite in contrast to MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in MMP-1 the C-terminal hemopexin domain does not interact with TIMP-1. The integrity of the catalytic domain of MMP-1 and its ability to bind Zn²⁺ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1 (Vallon et al. 1997). Therefore, the enzyme is also inhibited by chelators of divalent cations like EDTA or o-phenantroline.

Swiss-Prot link P03956: MMP-1 (human)
AfCS Signalling Gateway link A001461: MMP-1 (mouse)

Further Categories Containing This Product:

Natural Proteins • Enzymes

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