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Magic RedTM Detection of Cathepsins

Elevated cathepsin enzyme activity in serum or the extracellular matrix often signifies a number of gross pathological conditions. Cathepsin-mediated diseases include: Alzheimer's; numerous types of cancer; autoimmune related diseases like arthritis; and the accelerated breakdown of bone structure seen with osteoporosis1-2. Up-regulated cathepsin B and L activity has been linked to several types of cancer. These include cancer of the colon, pancreas, ovaries, breast, lung, and skin (melanoma)3-6.

Cathepsins are usually characterized as members of the lysosomal cysteine protease (active site) family7 and the cathepsin family name has been synonymous with lysosomal proteolytic enzymes1. In actuality, the cathepsin family also contains members of the serine protease (cathepsin A,G) and aspartic protease (cathepsin D,E) families as well.

The Magic Redd detection kits utilize the fluorophore, cresyl violet. When bi-substituted via amide linkage to two cathepsin target sequence peptides {such as (Arginine-Arginine)2}, the cresyl violet leaving group is non-fluorescent8. Following enzymatic cleavage at one or both arginine (R) amide linkage sites, the mono and non-substituted cresyl violet fluorophores generate red fluorescence when excited at 550-590 nm. B-Bridge also offers Magic Redd detection kits for caspase activity.

 

A B

Figure 3. Detection of intracellular cathepsin L activity in Jurkat cells using (z-FR)2-MR-Cathepsin fluorogenic substrate. Intracellular localization of the hydrolyzed (fluorescent) Magic Redd product was detected on a Nikon Eclipse E800 photomicroscope using a 510 560 nm excitation filter and a 570 620 nm emission/barrier filter at 400X (Photo A). Photo B shows the corresponding differential interference contrast (DIC) image of the cells.

References:

1. Buhling F, Fengler A, Brandt W, Welte T, Ansorge S, Nagler, DK. Review: novel cysteine proteases of the papain family. Adv Exp Med Biol 2000; 477:241-254. 
2. Gerber A, Welte T, Ansorge S, Buhling F. Expression of cathepsin B and L in human lung epithelial cells is regulated by cytokines. Adv Exp Med Biol 2000; 477:287-292.
3. Kas J, Werle B, Lah T, Brunner N. Cysteine proteinases and their inhibitors in extracellular fluids: markers for diagnosis and prognosis in cancer. Int J Biol Markers 2000; 15:84-89. 
4. Bank U, Kruger S, Langner J, Roessner A. Review: peptidases and peptidase inhibitors in the pathogenesis of diseases. Adv Exp Med Biol 2000; 477:349-378.
5. Guinec N, Fumeron VD, Pagano M. In vitro study of basement membrane degradation by the cysteine proteinases, cathepsin B, B-like, and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin. Biol Chem Hoppe-Seyler 1993; 374:1135-1146.
6. Frosch BA, Berquin I, Emmert-Buck MR, Moin K, Sloane BF. Molecular regulation, membrane association and secretion of tumor cathepsin B. APMIS 1999; 107:28-37.
7. Leung-Toung R, Li W, Tam TF, Kariman K. Thiol-dependent enzymes and their inhibitors: a review. Curr Med Chem 2002; 9:979-1002.
8. Van Noorden CJF, Boonacker E, Bissell ER, Meijer AJ, Van Marle J, Smith RE. Ala-Pro-cresyl violet, a synthetic fluorogenic substrate for the analysis of kinetic parameters of dipeptidyl peptidase IV (CD26) in individual living rat hepatocytes. Anal Biochem 1997; 252:71-77.

 

 


 

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