title: The Caco-2 cell monolayers as an intestinal metabolism model: metabolism of dipeptide Phe-Pro.
abstract: The metabolism of Phe-Pro was investigated in Caco-2 cell monolayers, a model of small intestinal epithelium. The results indicate that the majority of Phe-Pro was hydrolyzed during passage from the apical (AP) to basolateral (BL) side. The enzyme responsible for the hydrolysis is prolidase, a cytosolic enzyme. Through kinetic studies of a supernatant enzyme preparation, a Km of 30.4 microM and Vmax of 38.9 nmol/min per mg of protein were obtained. The enzyme catalyzed hydrolysis was inhibited by proline (66%), Zn+ (86%), Cu++ (100%), Fe (100%), PCMB (89%), and captopril (66%), but not by leucine. We also studied the transcellular transport of Phe-Pro by measuring the amount of Phe in the receiver media. In the presence of a proton gradient (AP pH6, BL pH7.4), the appearance rate of Phe in the BL media after Phe-Pro was loaded apically was at least 100 times faster than that in the AP media after Phe-Pro was loaded basolaterally. The former is also higher than the appearance rate of Phe without a transepithelial proton gradient (pH 6-pH 6) or against a proton gradient (pH7.4-pH6). The rate of appearance of Phe in the BL media (pH7.4) after Phe-Pro was loaded on the AP side (pH 6) was decreased by the presence in the AP media of proline (42%), leucine (40%), and captopril (17%), but not by Zn++. In conclusion, the transmembrane uptake of Phe-Pro is dependent on a proton gradient, and the intracellular metabolism of Phe-Pro is complete via hydrolysis by prolidase.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman 99164-6510.
authors.forename: M
authors.initials: M
authors.lastname: Hu
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Tran
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: G
authors.initials: G
authors.lastname: Leonardo
mesh_terms: D002455:Cell Division / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D015194:Diffusion Chambers, Culture; D004150:Dipeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007408:Intestinal Absorption / Q000502:physiology*; D007422:Intestines / Q000201:enzymology; D008954:Models, Biological; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D014407:Tumor Cells, Cultured
chemical_list: D004151:Dipeptides; C088360:phenylalanylproline; D004150:Dipeptidases; C022449:proline dipeptidase