CycLex Rho-kinase Assay Kit
| Code | Size | Price |
|---|
| MBL-CY-1160 | 96 Assays | £577.00 |
Quantity:
Prices exclude any Taxes / VAT
Overview
Regulatory Status: RUO
Shipping:
4°C
Storage:
4°C
Documents
Further Information
Applications:
Other - 1) Monitoring the purification of Rho-kinase or DMPK family kinase.
2) Screening inhibitors or activators of Rho-kinase or DMPK family kinase.
3) Detecting the effects of pharmacological agents on Rho-kinase or DMPK family kinase.
Background:
The small GTPase Rho regulates formation of focal adhesions and stress fibers of fibroblasts, as well as adhesion and aggregation of platelets and lymphocytes by shuttling between the inactive GDP-bound form and the active GTP-bound form. Rho is also essential in cytokinesis and plays a role in transcriptional activation by serum response factor. Ishizaki et al. (1996) identified the protein serine/threonine kinase ROCK1 (Rho-kinase beta), which they called p160-ROCK, which is activated when bound to the GTP-bound form of RhoA. Fujisawa et al. (1996) localized the Rho-binding domain of ROCK1 to a region between residues 934-1015.
ROCK2 (Rho-kinase alpha) is a serine/threonine kinase that regulates cytokinesis, smooth muscle contraction, the formation of actin stress fibers and focal adhesions, and the activation of the c-fos serum response element. ROCK2, which is an isozyme of ROCK1, is a target for the small GTPase Rho. Nakamura et al. (2001) studied the role of Rho in the migration of corneal epithelial cells in rabbit. They detected both ROCK1 and ROCK2 in the corneal epithelium at protein and mRNA levels. They found that exoenzyme C3, a Rho inhibitor, inhibits corneal epithelial migration in a dose-dependent manner and prevents the stimulatory effect of the Rho activator lysophosphatidic acid (LPA). Both cytochalasin B, an inhibitor of actin filament assembly, and ML7, an inhibitor of myosin light chain kinase, also prevent LPA stimulation of epithelial migration. The authors suggested that Rho mediates corneal epithelial migration in response to external stimuli by regulating the organization of the actin cytoskeleton.
Rao et al. (2001) investigated the role of Rho-kinase in the modulation of aqueous humor outflow facility. The treatment of human trabecular meshwork and canal of Schlemm cells with a Rho-kinase-specific inhibitor led to significant but reversible changes in cell shape and decreased actin stress fibers, focal adhesions, and protein phosphotyrosine staining. Based on the Rho-kinase inhibitor-induced changes in myosin light chain phosphorylation and actomyosin organization, the authors suggested that cellular relaxation and loss of cell-substratum adhesions in the human trabecular meshwork and canal of Schlemm cells could result in either increased paracellular fluid flow across the canal of Schlemm or altered flow pathway through the juxtacanalicular tissue, thereby lowering resistance to outflow. They suggested Rho-kinase as a potential target for the development of drugs to modulate intraocular pressure in glaucoma patients.
Description:
The CycLex Research Product CycLex Rho-kinase Assay kit is primarily designed to measure the activities of purified Rho-kinase or DMPK for the rapid and sensitive evaluation of inhibitors or activators. The phospho-specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phospho-threonine 696 residue in MBS/MYPT1, which is phosphorylated by Rho-kinase or DMPK (Myotonic dystrophy protein kinase) family kinases. Additionally, column fractions of cultured primary cell, cell line, or tissue homogenate can be assayed for Rho-kinase family activity with the CycLex Research Product CycLex Rho-kinase Assay kit if the appropriate dose of Rho-kinase specific inhibitor e.g. Y-27632 or HA-1077 is used.
Kit Components:
Microplate (coated with recombinant MBS C-terminus(758-1032 a.a.) as Rho-kinase substrate), 10?~Wash Buffer, Kinase Buffer, 20?~ATP, HRP conjugated Detection Antibody, Substrate Reagent, Stop Solution
Target:
Rho
References
1. Ishizaki, T., Mackawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakisuka, A., Morii, N. and Narumiya, S. EMBO J. 15: 1885-1893, 1996.
2. Fujisawa, K., Fujita, A., Ishizaki, T., Saito, Y. and Narumiya, S. J. Biol. Chem. 271: 23022-23028, 1996.
3. Nakamura, M., Nagano, T., Chikama, T. and Nishida, T. Invest. Ophthal. Vis. Sci. 42: 941-947, 2001.
4. Rao, P. V., Deng, P.-F., Kumar, J. and Epstein, D. L. Invest. Ophthal. Vis. Sci. 42: 1029-1037, 2001.
5. Muranyi, A, Zhang R, Liu F, Hirano K, Ito M, Epstein HF, Hartshorne DJ. Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Lett. 493(2-3):80-4, 2001.
6. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, Nakano T. Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett. 475(3):197-200, 2000.
7. Hamaguchi T, Ito M, Feng J, Seko T, Koyama M, Machida H, Takase K, Amano M, Kaibuchi K, Hartshorne DJ, Nakano T. Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem. Biophys. Res. Commun. 274(3):825-30, 2000.
8. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc. Natl. Acad. Sci. USA. 98(5):2419-24, 2001.
9. Niiro N and Ikebe M. ZIPer-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem. 276(31):29567-74, 2001.
10. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) Science. 273(5272):245-8, 1996.
11. Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.
J Cell Biol. 147(5):1023-38, 1999.
12. Nagumo H, Sasaki Y, Ono Y, Okamoto H, Seto M, Takuwa Y. Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am. J. Physiol. Cell Physiol. 278(1):C57-65, 2000.
13. Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15(9):2208-16, 1996.