A new type of fluorogenic substrates for determination of cellular dipeptidyl peptidase IV (DP IV/CD26) activity

The stability of cell associated fluorescence is an essentiell requirement for measurements of cellular enzymatic activity via enzyme catalyzed liberation of fluorophores. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize nonfluorescent dipeptidyl peptidase IV (DP TV) substrates Xaa-Pro-R110-Y allowing the stable covalent binding of the enzymaticaliy released fluorescent R110-Y on cells. All compounds have been characterized as substrates of isolated DP IV with k(cat)/K-m values of about 10(6) M-1.S-1. The hydrophobicity of the residue Y affects the affinity of the substrate to the catalytic site of DP TV. The compounds are characterized as sensitive substrates of cell surface associated DP nr 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.