Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay.
A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.
Jean
F
F
J. A. De Sève Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montréal (Affiliated with l'Université de Montréal), Québec, Canada.
Basak
A
A
Chrétien
M
M
Lazure
C
C
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000032:analysis*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011874:Radiometry / Q000379:methods*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D007457:Iodine Radioisotopes; D010455:Peptides; D010450:Endopeptidases