RESUMEN
The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the Sâ¢tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/química , Duodeno/metabolismo , Regulación de la Expresión Génica , Hígado/metabolismo , Fragmentos de Péptidos/normas , Ribonucleasa Pancreática/normas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/normas , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/normas , Duodeno/química , Células HEK293 , Humanos , Immunoblotting/métodos , Immunoblotting/normas , Hígado/química , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/normas , Proteínas de Neoplasias/biosíntesis , Fragmentos de Péptidos/química , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Ribonucleasa Pancreática/químicaRESUMEN
The pI value of rat erythrocyte 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) was determined to be 5.9 at 10 degrees C by isoelectric focusing in a horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). In this study, ribonuclease A-glutathione mixed disulfides (RNase-SG's) (T. Ubuka et al. (1986) J. Chromatogr., 363, 431-437) were used as pI standards. A mixture of RNase-SG was prepared by reducing bovine pancreatic ribonuclease A (RNase) with dithiothreitol and then treating the reduced RNase with oxidized glutathione. The mixture was composed of eight species which contained 1 (RNase-SG1) to 8 (RNase-SG8) mol of glutathione per mole of RNase, and the pI values of these species were determined under conditions minimizing the effect of carbon dioxide. The newly determined pI values of RNase-SG1 through RNase-SG8 were 8.8, 8.2, 7.7, 7.3, 6.9, 6.4, 5.8, and 5.3, respectively. The average change in pI values of these disulfides was 0.50 pH unit per mole of the bound glutathione per mole of RNase. The RNase-SG mixture was stable in acidic solutions and could be stored at 4 degrees C as well as at -20 degrees C with little change for at least 1 year. Thus, the mixture is shown to be an excellent standard for the determination of pI values of proteins by isoelectric focusing in the wide range of pI value.