Cytochrome P450 purification

Multiplicity of Cytochrome P450, Purification, and Reconstitution of Cytochrome P450 Activity. Even before appreciable purification of CYP had been accomplished, it was apparent from indirect evidence that mammalian liver cells contained more than one CYP enzyme. Subsequent direct evidence on the multiplicity of CYPs included the separation and purification of CYP isozymes, distinguished from each other by chromatographic behavior, immunologic specificity, and/or substrate specificity after reconstitution and separation of distinct polypeptides by sodium... 

Buhler, D.R. and J.L. Wang-Buhler. Rainbow trout cytochrome P450s purification, molecular aspects, metabolic activity, induction and role in environmental monitoring. Comp. Biochem. Physiol. C 121 107-137, 1998. 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Sasaki M, A Akahira, L-i Oshiman, T Tsuchid, Y Matsumura (2005) Purification of cytochrome P450 and ferredoxin involved in bisphenol. A degradation from Sphingomonas sp. stain AOl. Appl Environ Microbiol 71 8024-8030. 

GILLAM, E.M.J., BABA, T, KIM, B-R., OHMORI, S., GUENGERICH, F.P., Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme, Arch. Biochem. Biophys., 1993, 305, 123-131. 

Haining RL, Hunter AP, Veronese ME, et al. Allelic variants of human cytochrome P450 2C9 baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms. Arch Biochem Biophys 1996 333(2) 447 t58. 

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

Bergh AF, Strobel HW. 1992. Reconstitution ofthe brain mixed function oxidase system purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. J Neurochem 59 575-581. 

Bhamre S, Anandatheerathavarada HK, Shankar SK, Boyd MR, Ravindranath V. 1993. Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities. Arch Biochem Biophys 301 251-255. 

Dalet-Beluche 1, Boulenc X, Eabre G, Maurel P, Bonfils C (1992) Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. Cross reactivity with human forms. Eur J Biochem 204 641-648 Damaj Ml, Siu EC, Sellers EM, Tyndale RE, Martin BR (2007) Inhibition of nicotine metabolism by methoxysalen Pharmacokinetic and pharmacological studies in mice. J Pharmacol Exp Ther 320 250-257... 

Ohmori S, Horie T, Guengerich FP, Kiuchi M, Kitada M (1993b) Purification and characterization of two forms of hepatic microsomal cytochrome P450 from untreated cynomolgus monkeys. Arch Biochem Biophys 305 405 13... 

Gabriac B, Werck-Reichhart D, Teutsch H, Durst F (1991) Purification and immunochar-acterization of a plant cytochrome P450 the cinnamic acid 4-hydroxylase. Arch Biochem Biophys 288(l) 302-309... 

Mizutani M, Ohta D, Sato R (1993) Purification and characterization of a cytochrome P450 (rran -cinnamic acid 4-hydroxylase) from etiolated mung bean seedlings. Plant Cell Physiol 34 481-488... 

For proteins that are members of gene families, such as cytochrome P450, available structural models derived from X-ray crystallography and NMR data allow inference of the structure of newly discovered family members. The inference is based on the sequence variations derived from comparing the reference and new DNA sequences. While a high degree of computational power is needed, predictions based on this approach provide preliminary estimates of protein structure without a huge investment in expression, purification, and crystallization. A full structural elucidation requires several years for each protein, even if it can be crystallized. 

Matsuoka T, Miyakoshi S et al (1989) Purification and characterization of cytochrome P450 from Streptomyces carbophilus. Eur J Biochem 184 707-713... 

Arlotto MP, Greenway DJ, Parkinson A. Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7 alpha-hydroxylase activity. Arch Biochem Biophys 1989 270 441 457. 

Ponnamperuma, K. and Croteau, R., Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular, Arch. Biochem. Biophys., 329, 9-16, 1996. 

KOCHS, G., GRISEBACH, H., Phytoalexin synthesis in soybean purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase. Arch Biochem. Biophys., 1989,273,543-553. 

MEIJER, A.H., DE WAAL, A., VERPOORTE, R., Purification of cytochrome P450 enzyme geraniol 10 hydroxylase from cell cultures of Catharanthus roseus., J. Chromatog., 1993,35,237-249. 

Yang CS, Patten CJ, Ishizaki H, Yoo JSH (1991) Induction, purification, and characterization of cytochrome P450IIE. Methods Enzymol 206 595-603 Yuan R, Madani S, Wei XX et al. (2002) Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions. Drug Metab Dispos 30 1311-1319... 

Gillam, E. M. J., Guo, Z., and Guengerich, F. P., 1994, Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties, Arch. Biochem. Biophys. 312 59666. 

Meijer, A.H., De Wall, A. and Verpoorte, R. (1993a) Purification of the cytochrome P450 enzyme, geraniol 10-hydroxylase, from cell cultures of Catharanthus.. Chromatogr., 635, 237M9. 

Stadler, R. and Zenk, M.H. (1993) The purification and characterization of a unique cytochrome P450 enzyme from Berberis stolonifera plant cell cultures.. Biol. Chem., 268, 823-31. 

Halkier, B.A., Nielsen, H.L., Koch, B. and Muller, B.L. (1995) Purification and characterization of recombinant cytochrome P450, cyr expressed at high levels in Escherichia coli. Arch. Biochem. Biophys., 322, 369-77. 

Carbon monoxide inhibited the 6/3-. la-, and 16a-hydroxylation of testosterone by rat liver microsomes to different extents. A C0/02 ratio of 0.5 inhibited the la-, 6/i-, and 16a-hydroxylation reactions by 14%, 25%, and 36%, respectively, and the ratio of C0/02 needed for 50% inhibition of testosterone hydroxylation in the 16a-, 6/3-, and 7a-positions was 0.93, 1.54, and 2.36, respectively (36,48). Studies on the photochemical action spectrum revealed that CO inhibition of the three hydroxylation reactions was maximally reversed by monochromatic light at 450 nm, but there were differences in the shape of the photochemical reactivation spectra for the 6/3-, la-, and 16a-hydroxylation reactions (36,48). The data from our laboratory summarized above and at the First International Symposium on Microsomes and Drug Oxidation in 1968 pointed to multiple cytochromes P450 with different catalytic activities that were under separate regulatory control (36,45,46), and we indicated that the actual number of cytochromes that participate in the multiple hydroxylation reactions must await the solubilization and purification of the microsomal system (36). The use of different inducers of liver microsomal monooxygenases caused selective increases in the concentration of specific cytochromes P450 in fiver microsomes that greatly facilitated the isolation and purification of these hemoproteins. 

NADPH cytochrome P450 reductase, and a lipid fraction or phosphatidylcholine (51,52). The reconstituted hydroxylation system was then shown to metabolize a large number of drugs, carcinogens, and steroid hormones. These studies paved the way for subsequent studies on the purification and characterization of cytochrome P450. 

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