High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells.
A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.
Grimwood
B G
BG
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Tarentino
A L
AL
Plummer
T H
TH
D002268:Carboxypeptidases / Q000032:analysis*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000032:analysis; D004151:Dipeptides / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D008113:Liver Neoplasms / Q000201:enzymology*; D001121:Lysine Carboxypeptidase / Q000032:analysis* / Q000378:metabolism; D008832:Microchemistry; D014407:Tumor Cells, Cultured / Q000201:enzymology
D003470:Culture Media; D004151:Dipeptides; C028059:furylacryloylalanyllysine; D002268:Carboxypeptidases; D001121:Lysine Carboxypeptidase