Nov 24, 1989 - Samples of purified LPL, from the sources stated, were coated on to Falcon ... Financial support from A.R.F.C. and S.E.R.C. in support of this.
633rd MEETING, LONDON
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Table 1. lmmunoreactivities of lipoprotein Iipases revealed using an e.1.i.s.a.format Samples of purified LPL, from the sources stated, were coated on to Falcon assay plates ( 2 ,ug/ml in carbonate/bicarbonate buffer, pH 9.6). The reactivity of each sera was tested using appropriate enzyme-linked antibody conjugates. Key: - , no reactivity; + , + + , + + + , + + + + , reactivity (weakest to strongest). Antisera ~
~
~~
Chicken antirat cardiac
Mouse antirat cardiac
Mouse anti-
LPL sources
Rabbit antiovine cardiac
Bovine milk Ovine milk Ovine cardiac Rat cardiac Rat adipose Rat lung
++++ +++ + + ++ +
++++
++++
++++
enzyme from other sources, is the most effective antigen to react with any of the antisera produced. The data also confirm that the original observations of Al-Jafari & Cryer [3], that LpL was a component of the rat adipocyte plasma membrane using chicken anti-rat heart LPL can now be extended to include adipocyte plasma membranes Of chicken, ovine and porcine origins. Financial support from A.R.F.C. and S.E.R.C. in support of this work is gratefully acknowledged. I . Cryer, A. ( 1987) in Lipoprotein Lipase (Borensztajn,J., ed.),pp. 277-328, Evener Publishers Inc., Chicago 2. Cooper, D. A., Stein, J. C.. Strieleman, P. J. & Bensadoun, A. (1989)Biochim. Biophys. Acta 1008,92-101
-
-
+
bovine
-
-
-
-
-
-
+
+-
-
-
-
3. Al-Jafari, A. A. & Cryer, A. (1986) Biochern. J. 238,239-249 4. Parkin, S. M., Speake, B. K. & Robinson, D. S. ( I 982) Biorhem. J. 207,485-495 5. CrYer, A. Jones, H. M. (1978) Biochem. J . 1727 31 9-325 6. Voyta, J. C., Via, D. P., Kunnunen, P. K.. Sparrow, J. T., Gotto, A. M. & Smith, L. C. ( 1 985) J. Biol. Chem. 260,893-898 7. Laemmli, U.K. (1970)Nature (London)222, 680-688 8. Rechlin, M., Nisonoff, A. & Margoliash, E. (1970) J . Hiol. Chem. 245,947-954 9. Vaughan-Thomas, A. (1988)PhD Thesis, University of Wales 10. Belsham, G. J., Denton, R. M. & Tanner, M. J. ( 1980) Hiochem. J . 192,45
Received 2 1 November 1989
Quantification of cytochrome F450 gene expression in human tissues COLIN N. A. PALMER,* ELIZABETH A. SHEPHARDT and IAN R. PHILLIPS* *Department of Biochemistry, The Medical College o f s t Bartholomew S Hospital, University of London, Charterhouse Sqirare, London ECIM bBQ, U.K. and TDepartment of Biochemistry, University College London, Gower Street, London WClE bBT, U.K. Humans can metabolize an extremely wide range of potentially harmful foreign hydrophobic compounds including many drugs, environmental pollutants and chemical carcinogens. This ability is mainly due to the action of cytochromes 1’-450. These proteins constitute a large superfamily that has been classified into several families, the largest of which (P45011) has been further divided into seven subfamilies (IIA-IIG) [ 11. Variations in the structure or expression of cytochrome 1’-450 genes are thought to be responsible for many of the known inherited variations in the metabolism of therapeutic drugs [2].The best understood example is that of debrisoquine hydroxylation, in which the poor metabolizer phenotype is due to mutations that cause abnormal RNA rocessing, resulting in the production of an unstable mRNA 31. In addition to being subject to genetic variation, the expression of many cytochrome P-450 genes is influenced by environmental factors. Determination of the level of expression of cytochrome P-450 genes in human tissues will contribute greatly to our understanding of the molecular basis of interindividual variations in foreign compound metabolism. Few human cytochromes f - 4 5 0 have been purified to homogeneity.
p.
VOl. 18
However, cDNA clones for many different cytochromes 1’450 have been isolated by ourselves [4-81 and by other groups (reviewed in [9]),and these can be used to measure the expression of the corresponding genes at the mRNA level. Northern-blot hybridization with cDNA probes is often not specific enough to distinguish between closely related cytochrome P-450 mRNAs. Synthetic oligonucleotides, although highly specific, lack the sensitivity required for the quantification of low abundance mRNAs. To overcome these problems we have used a technique, based on RNAase protection [lo], which is highly specific, and can detect as few as 0.05 copies of mRNA per cell. A 32P-labelled ‘antisense’ RNA is hybridized in solution to total tissue RNA. Probe not protected by hybridization to its complementary mRNA is digested by RNAase. Protected probe is electrophoresed through a denaturing polyacrylamide gel, detected by autoradiography and quantified by scanning densitometry. Comparison with a standard curve of undigested radiolabelled probe permits the absolute quantification of the corresponding mRNA. Assays of known amounts of the mRNA, produced by transcription of a cDNA clone in vitro, confirmed the accuracy of the method. We have used this technique to measure the expression of several cytochrome P-450 genes in over 20 human livers. The P450IA2 gene displays a 30-fold interindividual variation in expression (from 0.2 to 6 mRNA molecules/cell) (Fig. l a ) . A similar variation is found for P450IIC (1-30 mRNAs/cell). Other cytochrome P-450 genes exhibit variations in expression ranging from 10-fold (0.2-2 mRNAs/cell) in the case of P4501IB, to 1000-fold (0.05-50 mRNAs/cell) for P450IIA.
BIOCHEMICAL SOClETY TRANSACTIONS
616 (b)
(a) a
b c d e f g ti i j k I
a b c d e
f
g
h
i
j
k
I
334 285
Fig. 1. RNAase protection assay of mRNAs coding for PP450IA2(a) and P-450 reductase (b) ( a )Full-length undigested probe (track a); probe protected after hybridization to 40 g
of tRNA (track b) or total RNA from different livers (tracks c-1); 8 h exposure. ( b ) Size markers (1 kb) ladder) (track a); full-length undigested probe (track b); probe protected after hybridization to 20 g of total RNA from different livers (tracks c-I); 48 h exposure. Numbers indicate sizes in nucleotides of full-length and protected probes.
In contrast, the expression of the cytochrome P-450 reductase gene [ l l ] varies by only 3-fold (1-3 mRNAs/cell) between different individuals (Fig. 1b). None of the cytochrome P-450 genes examined is coordinately regulated. The expression of some of them appears to be confined to the liver, whereas the cytochrome P-450 reductase gene is not subject to tissue-specific regulation. Our results demonstrate considerable interindividual variation in the level of expression of many cytochrome P450 genes and this may have important implications for a person’s ability to metabolize drugs and carcinogens. Many of the livers we have analysed have been phenotyped with respect to foreign compound metabolism. Therefore, it may be possible to establish correlations between the level of expression of individual cytochrome P-450 genes and particular metabolic activities, thus providing insights into the biological role of the gene products. We thank Dr Urs Meyer for some of the liver samples. This work was supported by grants from the C.R.C., M.R.C., and the Joint Research Board of St Bartholomew’s Hospital Medical College. 1. Nebert, D. W., Nelson, D. R., Adesnik, M., Coon, M. J.,
Estabrook, R. W., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Kemper, B., Levin, W., Phillips, I. R., Sato, R. & Waterman, M. R. ( 1989) DNA 8 , l - 13
2. Miners, J., Birkett, D. J., Drew, R. & McManus, M. (eds.) ( 1 988)
Microsornes and Drug Oxidations, Taylor and Francis, London 3. Gonzalez, F. J., Skoda, R., Kimura, S., Umeno, M., Zanger, U. M., Nebert, D. W., Gelboin, H. V., Hardwick, J. P. & Meyer, U. A. (1988) Nature (London)331,442-446 4. Phillips, I. R., Shephard, E. A,, Ashworth, A. & Rabin, B. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82,983-987
5. Phillips, I. R., Shephard, E. A., Griffith, C., Santisteban, I.,
6. 7. 8.
9.
10. 11.
Gammon, B. M., Hutter, A., Bell, D. R. & Forrest, L. A. (1988) in Microsornes and Drug Oxidations (Miners, J., Birkett, D. J., Drew, R. & McManus, J., eds.), pp. 89-95, Taylor and Francis, London Santisteban, I., Povey, S., Shephard, E. A. & Phillips, I. R. (1988)Ann. Hum. Genet. 52,129-135 Shephard, E. A., Phillips, I. R., Santisteban, I., Palmer, C. N. A. & Povey, S. ( 1989) Ann. Hum. Genet. 53,23-3 1 Griffith, C. J., Shephard, E. A., Palmer, C. N. A., Santisteban, I. & Phillips, I. R. ( 1989) in Cytochrome P-450.Biochemistry, Biophysics and Environmental Implications (Schuster, I., ed.), pp. 548-55 1, Elsevier Biomedical Press, Amsterdam Gonzalez, F. J. ( 1989) Pharmacol. Rev. 40,243-288 Zinn, K., di Maio, D. & Maniatis, T. (1984) Cell (Cambridge, Mass.) 34,865-879 Shephard, E. A., Phillips, I. R., Santisteban, I., West, L. F., Palmer, C. N. A., Ashworth, A. & Povey, S. ( 1 989) Ann. Hum. Genet. 53.291-301
Received 24 November 1989
1990
