Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection.
The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.
Ohno
M
M
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Kai
M
M
Ohkura
Y
Y
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D008297:Male; D015260:Neprilysin / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D015260:Neprilysin