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 essential 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 IV) substrates Xaa-Pro-R110-Y allowing the stable covalent binding of the enzymatically released fluorescent R110-Y on cells. All compounds have been characterized as substrates of isolated DP IV with kcat/Km 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 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.
Lorey
S
S
Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Germany.
Faust
J
J
Bühling
F
F
Ansorge
S
S
Neubert
K
K
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism*; D018819:Dipeptidyl Peptidase 4 / Q000032:analysis / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D012235:Rhodamines / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D020298:U937 Cells / Q000201:enzymology
D000596:Amino Acids; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D012235:Rhodamines; C049025:rhodamine 110; D011392:Proline; D018819:Dipeptidyl Peptidase 4