Abstract
The stability of cell associated fluorescence is an essentiell requirement for measurements of cellular enzymatic activity via enzyme catalyzed liberation of fluorophores. Rhodamine 110 (Rl10), a highly fluorescent Athene dye, was used to synthesize nonfluorescent dipeptidyl peptidase IV (DP IV) substrates Xaa-Pro-R110-Y allowing the stable covalent binding of the enzymatically released fluorescent R110- on cells. All compounds have been characterized as substrates of isolated DP IV with kcat/Km values of about 106 M–1. s–1. The hydrophobicity of the residue affects the affinity of the substrate to the catalytic site of DP IV. The compounds are characterized as sensitive substrates of cell surface associated DP IV of DP IV rich U-937 cells. The binding of the enzymatically released R110-Y on cells results in a stable cellular fluorescence. This way, the quantitative determination of cell surface associated DP IV activity is possible.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Ansorge, S., Bühling, F., Hoffmann, T., Kähne, T., Neubert, K., and Reinhold, D., 1995, In Dipeptidyl peptidase VI (CD26) in Metabolism and the Immune Response (B. Fleischer, ed), Springer-Verlag, Heidelberg, pp. 163–184.
Bühling, F., Junker, U., Neubert, K., Jäger, L., and Ansorge, S., 1995, Functional role of CD26 on human lymphocytes. Immunol. Lett. 45: 47–51.
Heins, J., Welker, P., Schönlein, C., Born, I., Hardrodt, B., Neubert, K., Tsuru, D. and Barth, A., 1988, Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline specificendopeptidase from Flavobacterium meningosepticum. Biochim. Biophys. Acta 954: 161–169.
Kähne, T., Neubert, K., Faust, J., and Ansorge, S., 1998, Early phosphorylation events induced by DP IV/CD26-specific inhibitors. Cell. Immunol. 189: 60–66.
Leytus, S.P., Melhado, L.L., and Mangel, W.F., 1983, Rhodamine-basedcompounds as fluorogenic substrates for serine proteinases. Biochem. J. 209: 299–307.
Lorey, S., Faust, J., Bühling, F., Ansorge, S., and Neubert, K., 1997, New fluorogenic Dipeptidyl peptidase IV/CD26 substrates and inhibitors. Adv. Exp. Med. Biol. 241: 157–160.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Kluwer Academic Publishers
About this chapter
Cite this chapter
Lorey, S., Faust, J., Bühling, F., Ansorge, S., Neubert, K. (2002). A New Type of Fluorogenic Substrates for Determination of Cellular Dipeptidyl Peptidese IV (DP IV/CD26) Activity. In: Langner, J., Ansorge, S. (eds) Cellular Peptidases in Immune Functions and Diseases 2. Advances in Experimental Medicine and Biology, vol 477. Springer, Boston, MA. https://doi.org/10.1007/0-306-46826-3_11
Download citation
DOI: https://doi.org/10.1007/0-306-46826-3_11
Publisher Name: Springer, Boston, MA
Print ISBN: 978-0-306-46383-9
Online ISBN: 978-0-306-46826-1
eBook Packages: Springer Book Archive