Improved determination of bovine glutaminyl cyclase activity using precolumn derivatization and reversed-phase high-performance liquid chromatography with ultraviolet detection

摘要

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of L-pyroglutamic acid β-naphthylamide at 235 nm, enzymatically formed from the substrate L-glutaminyl-β-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0–8.5. The K_m and V_(max) values were 100.2±2.9 μM and 332±21.7 pmol/(h μg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.