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Furin |
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Substrate Specificity:
Description:
Furin is a ubiquitous subtilisin-like proprotein convertase. It is the major processing enzyme of the secretory pathway and is localized in the trans-golgi network (1,2). Substrates of Furin include blood clotting factors, serum proteins and growth factor receptors such as the insulin-like growth factor receptor (3). The minimal cleavage site is Arg-X-X-Arg'. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg'. An additional arginine at the P6 position appears to enhance cleavage (4). Furin is inhibited by EGTA, α1- Antitrypsin Portland (5) and polyarginine compounds (6).
Source:
Isolated from Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus carrying truncated human furin (kindly provided by R. Fuller) (3).
Enzyme Properties
Specificity: The minimal cleavage site is Arg-X-X-Arg. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg.
Molecular Weight:
Theoretical: 52.7 kDa and Apparent: 57 kDa
Reaction & Storage Conditions
Unit Definition:
One unit is defined as the amount of Furin that will release 1 pmol of AMC from the fluorogenic peptide BOC-RVRR-AMC (Bachem #I-1645) in one minute (1 pmol of AMC/min) at 30°C.
Unit Assay Conditions: 100 mM HEPES (pH 7.5 @ 25°C), 0.5% Triton X-100, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 100 µM B0C-RVRR-AMC and enzyme in a 100 µl volume at 30°C.
Fusion Protein Digestion: One unit will cut 25 μg test substrate to 95% completion in 6 hours or less, while 0.5 units will cut 25 μg of test substrate to 95% completion in 16 hours or less.
Fusion Protein Digestion Conditions: Furin is added to 25 μg of an MBP fusion protein testsubstrate, MBP-ΔSal. The reaction is carried out in 25 ul, 100 mM Hepes (pH 7.5 @25°C), 0.5%Triton X-100, 1mM CaCl2, 1mM 2-mercaptoethanol at 25°C
Concentration:
2,000 units/ml
Storage Conditions:
10 mM MES
1 mM CaCl2
50% Glycerol
pH 7.0 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Both Furin and Onchocerca volvulus Blisterase will cleave peptide substrates with the sequence, Arg-X- (Lys/Arg)-Arg. However, the ability of either enzyme to cleave a particular protein substrate depends on its tertiary structure as well as on the amino acids immediately surrounding the cleavage site (7).
References
- van den Ouweland, A.M.W. et al. (1990) Nucl. Acids Res., 18, 664.
- Steiner, D.F. (1998) Curr. Opin. Chem. Biol., 2, 31-39.
- Bravo, D.A. et al. (1994) J. Biol. Chem., 269, 25830-25837.
- Krysan, D.J. et al. (1999) J. Biol. Chem., 274, 23229-23234.
- Jean, F. et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 7293-7298.
- Cameron, A. et al. (2000) J. Biol. Chem., 275, 36741-36749.
- Poole, C.B. et al. (2003) J. Biol. Chem., 278, 36183-36190.
Companion Products
Enterokinase, light chain
Factor Xa Protease
Genenase I
Proteinase K
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