Identification of 17--Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactiv
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- 作者：Ute M. Kent ; Hsia-lien Lin ; Danielle E. Mills ; Kelly A. Regal ; and Paul F. Hollenberg
- 刊名：Chemical Research in Toxicology
- 出版年：2006
- 出版时间：February 2006
- 年：2006
- 卷：19
- 期：2
- 页码：279 - 287
- 全文大小：155K
- 年卷期：v.19,no.2(February 2006)
- ISSN：1520-5010
文摘
The oral contraceptive 17-
fchars/alpha.gif" BORDER=0>-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochromeP450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus oneoxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separationof these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminalsequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) andPYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS)and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptideresulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolismof 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated differentreactive 17EE intermediates that were responsible for the inactivation and protein modification or theformation of GSH conjugates by these two enzymes.
fchars/alpha.gif" BORDER=0>-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochromeP450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus oneoxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separationof these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminalsequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) andPYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS)and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptideresulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolismof 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated differentreactive 17EE intermediates that were responsible for the inactivation and protein modification or theformation of GSH conjugates by these two enzymes.