Purification of a human liver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats.

Cytochrome P450 is a family of enzymes that catalyze reactions involved in the metabolism of drugs and other xenobiotics. These enzymes are therefore important in pharmacologic and toxicologic studies, and information on their abundances is of value in the process of scaling in vitro data to in vivo metabolic parameters. A meta-analysis was applied to data on the abundance of human hepatic cytochrome P450 enzymes in Caucasian adult livers (50 studies). Despite variations in the methods used to measure the abundance of enzymes, agreement between the studies in 26 different laboratories was generally good. Nonetheless, some heterogeneity was detected (Higgins and Thompson heterogeneity test). More importantly, large interindividual variability was observed in the collated data. Positive correlations between the expression levels of some cytochrome P450 enzymes were found in the abundance data, including the following pairs: CYP3A4/CYP3A5*1/*3 (Rs = 0.70, P < 0.0001, n = 52), CYP3A4/CYP2C8 (Rs = 0.68, P < 0.0001, n = 134), CYP3A4/CYP2C9 (Rs = 0.55, P < 0.0001, n = 71), and CYP2C8/CYP2C9 (Rs = 0.55, P < 0.0001, n = 99). These correlations can be used to demonstrate common genetic transcriptional mechanisms.

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“…Studies expressing abundance in relative units (Wrighton et al, 1987;Wrighton et al, 1993) were also used.…”

Section: Resultsmentioning

confidence: 99%

Expression of Hepatic Drug-Metabolizing Cytochrome P450 Enzymes and Their Intercorrelations: A Meta-Analysis

2014

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“…Microsomes were prepared and stored as previously described ( 18,30,31 ). Protein concentration was determined colorimetrically as previously described ( 18,30,31 (33). Positive phage were rescreened by use of this probe.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was isolated from specimens by guanidinium thiocyanate-phenol-chloroform extraction (34). The RNA (30 Mug) was fractionated by electrophoresis in a formaldehyde denaturing agarose gel (34) and transferred to nitrocellulose by capillary diffusion and then baked at 80'C under vacuum for 2 h. Prehybridization and hybridization with the 32P-radiolabeled CYP3A7 cDNA were as described previously (35). The size of the CYP3A7 hybridizing mRNA band was estimated by linear interpolation of 28S (4.9-kb) and 18S ( 1.95-kb) RNA standards.…”

Section: Methodsmentioning

confidence: 99%

Identification of the fetal liver cytochrome CYP3A7 in human endometrium and placenta.

Schuetz¹,

Kauma²,

Guzelian³

1993

J. Clin. Invest.

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104

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Placenta and endometrium carry out steroidogenic biotransformation reactions such as 6-ft-hydroxylation of cortisol, a reaction characteristic of the dominant family of cytochromes P450 in human liver, CYP3A. To investigate the possible role in these extrahepatic tissues of the CYP3A microsomal hemoproteins, we analyzed placental and endometrial microsomes on Western blots developed with an anti-human CYP3A antibody. We found an immunoreactive 51,500 D protein that migrated between CYP3A3 (HLp) and CYP3A5 (HLp2) identical with CYP3A7 (HFLa). CYP3A7, a form found prominently in human fetal liver microsomes, was first isolated as a liver 16-adehydroepiandrosterone-sulfate hydroxylase. Northern blot analysis of total RNA isolated from placenta or from endometrium demonstrated a single band that cross-hybridized with a CYP3A7 cDNA. Amplification of the same RNA samples with the use of primers specific for CYP3A7, produced a 552-bp segment that had the predicted size and the same DNA sequence as does liver CYP3A7 cDNA. Hybridizable endometrial CYP3A7 mRNA was detected more frequently (six of seven samples) and in higher amounts (-12-fold higher) in pregnant compared with nonpregnant women (4 of 12 samples). In addition, during the secretory phase of the menstrual cycle CYP3A7 expression was sixfold higher than in the one sample from the proliferative phase that had detectable CYP3A7 mRNA. Moreover, the amounts of placental and endometrial CYP3A7 mRNA and protein increased substantially from the first to the second trimester of pregnancy. We conclude that placenta and endometrium express the same P450 as is found in fetal liver. These tissues represent a previously unrecognized and quantitatively important site for 6-ft-hydroxylation and 16-a-hydroxylation of specific steroid precursors, possibly for protection of the fetus from the toxic effects of endogenous steroids and foreign substrates. (J. Clin. Invest. 1993.

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“…Guengerich et al, 1986). (References to the data for each of the P450s in A are 1A2: Distlerath et al 1985;2A6: Yun et al, 1991;2C8: Wang et al, 1980;Wrighton et al, 1987b;Lasker et al, 1998;2C9: Shimada et al, 1986;Kawano et al, 1987;Lasker et al, 1987Lasker et al, , 1998Komori et al, 1988;Sandhu et al, 1993;2C19: Lasker et al, 19872C19: Lasker et al, , 19982D6: Gut et al, 19842D6: Gut et al, , 1986Dislerath et al, 1985;2E1: Lasker et al, 1987;Wrighton et al, 1987a;3A4: Wang et al, 1983;Watkins et al, 1985;Guengerich et al, 1986;Kawano et al, 1987;Komori et al, 1988;Lin et al, 2002 …”

Section: Discussionmentioning

confidence: 99%

Disparity in Holoprotein/Apoprotein Ratios of Different Standards Used for Immunoquantification of Hepatic Cytochrome P450 Enzymes

Perrett

Barter

Jones³

et al. 2007

Drug Metab Dispos

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ABSTRACT:An analysis of reported hepatic abundances of CYP3A4 and 3A5 indicated that values determined by immunoquantification using commercially available, unpurified recombinant enzymes as standards are significantly lower than those determined using purified enzymes or human liver microsomes characterized with lysosomal peptides (CYP3A4: mean 45 versus 121 pmol/mg protein, p < 0.01; CYP3A5: mean 28 versus 83 pmol/mg protein, p < 0.05). When immunoquantifying cytochromes P450 (P450s), it is assumed that the holoprotein (holo)/apoprotein ratio is the same in the samples and the standard. Estimates of holo/apoprotein ratios from data reported for a range of P450s purified from human liver and noncommercial recombinant systems indicated less than complete and variable heme coupling dependent on enzyme and system.

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Purification of a human liver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats.

Cited by 52 publications

References 36 publications

Expression of Hepatic Drug-Metabolizing Cytochrome P450 Enzymes and Their Intercorrelations: A Meta-Analysis

Expression of Hepatic Drug-Metabolizing Cytochrome P450 Enzymes and Their Intercorrelations: A Meta-Analysis

Identification of the fetal liver cytochrome CYP3A7 in human endometrium and placenta.

Disparity in Holoprotein/Apoprotein Ratios of Different Standards Used for Immunoquantification of Hepatic Cytochrome P450 Enzymes

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