Characterization of New Fluorogenic Substrates for the Rapid and Sensitive Assay of Cathepsin E and Cathepsin D
-
- Yasuda Yoshiyuki
- Departments of Pharmacology Kyushu University Faculty of Dentistry Departments of Conservative Dentistry II, Kyushu University Faculty of Dentistry
-
- Kageyama Takashi
- Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University
-
- Akamine Akifumi
- Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University
-
- Shibata Masahiro
- Department of Cell Biology and Anatomy, Osaka University Medical School
-
- Kominami Eiki
- Department of Biochemistry, Juntendo University School of Medicine
-
- Uchiyama Yasuo
- Department of Cell Biology and Anatomy, Osaka University Medical School
-
- Yamamoto Kenji
- Departments of Conservative Dentistry II, Kyushu University Faculty of Dentistry
書誌事項
- タイトル別名
-
- Characterization of New Fluorogenic Substrates for the Rapid and Sensitive Assay of Cathepsin E and Cathepin D.
この論文をさがす
説明
Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2, 4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km=10.9 μM-1•s-1 for cathepsin E and 15.6 μM-1•s-1 for cathepsin D). A very acidic pH optimum of 4.0 was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp) γ-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km=12.2 μM-1•s-1 for cathepsin E and 16.3 μM-1•s-1 for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.
収録刊行物
-
- The Journal of Biochemistry
-
The Journal of Biochemistry 125 (6), 1137-1143, 1999
社団法人 日本生化学会