Abstract
We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [3H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.
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- Althaus F. R., Sinclair J. F., Sinclair P., Meyers U. A. Drug-mediated induction of cytochrome(s) P-450 and drug metabolism in cultured hepatocytes maintained in chemically defined medium. J Biol Chem. 1979 Mar 25;254(6):2148–2153. [PubMed] [Google Scholar]
- Bar-Nun S., Kreibich G., Adesnik M., Alterman L., Negishi M., Sabatini D. D. Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 1980 Feb;77(2):965–969. doi: 10.1073/pnas.77.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bhat K. S., Padmanaban G. Cytochrome P-450 synthesis in vivo and in a cell-free system from rat liver. FEBS Lett. 1978 May 15;89(2):337–340. doi: 10.1016/0014-5793(78)80250-6. [DOI] [PubMed] [Google Scholar]
- Bissell D. M., Guzelian P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci. 1980;349:85–98. doi: 10.1111/j.1749-6632.1980.tb29518.x. [DOI] [PubMed] [Google Scholar]
- Bissell D. M., Hammaker L. E. Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity. Arch Biochem Biophys. 1976 Sep;176(1):91–102. doi: 10.1016/0003-9861(76)90144-2. [DOI] [PubMed] [Google Scholar]
- Brunda M. J., Minden P., Sharpton T. R., McClatchy J. K., Farr R. S. Precipitation of radiolabeled antigen-antibody complexes with protein A-containing Staphylococcus aureus. J Immunol. 1977 Jul;119(1):193–198. [PubMed] [Google Scholar]
- Craft J. A., Cooper M. B., Estall M. R., Rabin B. R. The biosynthesis of cytochrome P450 by rough endoplasmic reticulum in vitro. A significant proportion of newly-biosynthesised cytochrome P450 is resistant to proteolytic digestion in intact vesicles. FEBS Lett. 1979 Feb 15;98(2):403–407. doi: 10.1016/0014-5793(79)80227-6. [DOI] [PubMed] [Google Scholar]
- Craft J. A., Cooper M. B., Estall M. R., Rees D. E., Rabin B. R. The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450. Eur J Biochem. 1979 May 15;96(2):379–391. doi: 10.1111/j.1432-1033.1979.tb13050.x. [DOI] [PubMed] [Google Scholar]
- Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
- Elshourbagy N. A., Guzelian P. S. Separation, purification, and characterization of a novel form of hepatic cytochrome P-450 from rats treated with pregnenolone-16 alpha-carbonitrile. J Biol Chem. 1980 Feb 25;255(4):1279–1285. [PubMed] [Google Scholar]
- Fahl W. E., Michalopoulos G., Sattler G. L., Jefcoate C. R., Pitot H. C. Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes. Arch Biochem Biophys. 1979 Jan;192(1):61–72. doi: 10.1016/0003-9861(79)90071-7. [DOI] [PubMed] [Google Scholar]
- Fry J. R., Wiebkin P., Bridges J. W. 7-Ethoxycoumarin O-deethylase induction by phenobarbitone and 1,2-benzanthracene in primary maintenance cultures of adult rat hepatocytes. Biochem Pharmacol. 1980 Feb 15;29(4):577–581. doi: 10.1016/0006-2952(80)90379-2. [DOI] [PubMed] [Google Scholar]
- Guzelian P. S., Bissell D. M., Meyer U. A. Drug metabolism in adult rat hepatocytes in primary monolayer culture. Gastroenterology. 1977 Jun;72(6):1232–1239. [PubMed] [Google Scholar]
- Haugen D. A., Coon M. J. Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with beta-naphthoflavone or phenobarbital. J Biol Chem. 1976 Mar 25;251(6):1817–1827. [PubMed] [Google Scholar]
- Ivarie R. D., Jones P. P. A rapid sensitive assay for specific protein synthesis in cells and in cell-free translations: use of Staphylococcus aureus as an adsorbent for immune complexes. Anal Biochem. 1979 Aug;97(1):24–35. doi: 10.1016/0003-2697(79)90322-1. [DOI] [PubMed] [Google Scholar]
- Kessler S. W. Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol. 1976 Nov;117(5 Pt 1):1482–1490. [PubMed] [Google Scholar]
- Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
- Kumar A., Padmanaban G. Studies on the synthesis of cytochrome P-450 and cytochrome P-448 in rat liver. J Biol Chem. 1980 Jan 25;255(2):522–525. [PubMed] [Google Scholar]
- Lu A. Y., West S. B. Multiplicity of mammalian microsomal cytochromes P-45. Pharmacol Rev. 1979 Dec;31(4):277–295. [PubMed] [Google Scholar]
- March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
- Michalopoulos G., Sattler C. A., Sattler G. L., Pitot H. C. Cytochrome P-450 induction by phenobarbital and 3-methylcholanthrene in primary cultures of hepatocytes. Science. 1976 Sep 3;193(4256):907–909. doi: 10.1126/science.948753. [DOI] [PubMed] [Google Scholar]
- Negishi M., Jensen N. M., Garcia G. S., Nebert D. W. Structural gene products of the murine Ah complex. Differences in ontogenesis and glucosamine incorporation between liver microsomal cytochromes P1-450 and P-448 induced by polycyclic aromatic compounds. Eur J Biochem. 1981 Apr;115(3):585–594. [PubMed] [Google Scholar]
- OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem. 1964 Jul;239:2379–2385. [PubMed] [Google Scholar]
- Ohlsson R. I., Lane C. D., Guengerich F. P. Synthesis and insertion, both in vivo and in vitro, of rat-liver cytochrome P-450 and epoxide hydratase into Xenopus laevis membranes. Eur J Biochem. 1981 Apr;115(2):367–373. doi: 10.1111/j.1432-1033.1981.tb05247.x. [DOI] [PubMed] [Google Scholar]
- Owens I. S., Nebert D. W. Aryl hydrocarbon hydroxylase induction in mammalian liver-derived cell cultures. Stimulation of "cytochrome P1-450-associated" enzyme activity by many inducing compounds. Mol Pharmacol. 1975 Jan;11(1):94–104. [PubMed] [Google Scholar]
- Rajamanickam C., Rao M. R., Padmanaban G. On the sequence of reactions leading to cytochrome P-450 synthesis-effect of drugs. J Biol Chem. 1975 Mar 25;250(6):2305–2310. [PubMed] [Google Scholar]
- Richards G. M. Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal Biochem. 1974 Feb;57(2):369–376. doi: 10.1016/0003-2697(74)90091-8. [DOI] [PubMed] [Google Scholar]
- Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
- Thomas P. E., Korzeniowski D., Ryan D., Levin W. Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes. Arch Biochem Biophys. 1979 Feb;192(2):524–532. doi: 10.1016/0003-9861(79)90122-x. [DOI] [PubMed] [Google Scholar]
- Toftgård R., Nilsen O. G., Ingelman-Sundberg M., Gustafsson J. A. Correlation between changes in enzymatic activities and induction of different forms of rat liver microsomal cytochrome P-450 after phenobarbital-, 3-methylcholanthrene- and 16 alpha-cyanopregnenolone treatment. Acta Pharmacol Toxicol (Copenh) 1980 May;46(5):353–361. doi: 10.1111/j.1600-0773.1980.tb02466.x. [DOI] [PubMed] [Google Scholar]