A New Type of Fluorogenic Substrates for Determination of Cellular Dipeptidyl Peptidese IV (DP IV/CD26) Activity

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 k_cat/K_m 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.