Martha V. Martin

Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat liver microsomes

Abstract A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems.

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature. 2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents. 3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.

Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase

Cytochrome P450 (P450) enzymes are important in the metabolism of steroids, vitamins, carcinogens, drugs and other compounds. Two of the commonly used assays in this field are the measurements of total P450 and NADPH–P450 reductase in biological preparations. A detailed protocol is presented for the measurement of P450 by its spectral properties, along with a protocol for measuring NADPH–P450 reductase by its NADPH–cytochrome c reduction activity. Each assay can be completed in 5–10 min. Detailed explanations for the rationale of particular sequences in the protocols are provided, along with potential confounding problems.

Purification of functional recombinant P450s from bacteria.

Publisher Summary This chapter discusses the methods for the purification of functional recombinant P450s from bacteria. A number of expression systems have been utilized to produce recombinant P450s, as discussed in this and an earlier volume. I Bacteria have considerable advantages because of the high levels of expression, ease of manipulation, and relatively low cost. While P450s can be studied within some of the other vector systems, purification is usually necessary for most studies with the enzymes produced in bacteria. In general, purification is relatively easy and considerably more efficient than from mammalian tissues Thes chapter focuses on recombinant human P450s expressed in Escherichia coli and, Salmonella typhimurium . The most direct means of monitoring P450 production in bacteria is to do direct difference spectroscopy.

Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation.

Nine forms of cytochrome P-450 have been purified to electrophoretic homogeneity from human-liver microsomes. These include the enzymes involved in debrisoquine 4-hydroxylation, phenacetin O-deethylation and mephenytoin 4-hydroxylation, three reactions which are characterized by genetic polymorphism in humans. Evidence for the involvement of the above enzymes comes from reconstituted immunochemical inhibition studies with human-liver microsomes. These and other lines of evidence are consonant with the view that different forms of cytochrome P-450 are involved in the three reactions. The debrisoquine 4-hydroxylase has been studied most extensively in terms of its substrate specificity. In addition, an analogous rat enzyme shows some homology and serves as a useful model. The use of antibodies raised to the rat-liver enzyme in immuno-inhibition studies with human-liver microsomes provides a means of determining the extent to which this enzyme participates in other reactions. Translation of rat-liver mRNA in vitro yields the intact debrisoquine 4-hydroxylase; studies with human mRNA suggest a lower frequency than in rats. The basis for impaired catalytic activity in phenotypically poor human metabolizers appears to be an altered enzyme in all three cases, as opposed to a decreased level of a single enzyme. Using antibody screening of fusion proteins expressed in a cDNA library, it has been possible to isolate cDNA probes for all three of these cytochromes P-450 for use in screening individuals and ultimately determining the basis of these polymorphisms.

Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2).

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH → FDR1 → Fdx4 → P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C–H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.

Development of Oxidative Stress by Cytochrome P450 Induction in Rodents Is Selective for Barbiturates and Related to Loss of Pyridine Nucleotide-dependent Protective Systems

Reactive oxygen species (ROS) and oxidative stress have been considered in a variety of disease models, and cytochrome P450 (P450) enzymes have been suggested to be a source of ROS. Induction of P450s by phenobarbital (PB), β-naphthoflavone (βNF), or clofibrate in a mouse model increased ROS parameters in the isolated liver microsomes, but isoniazid treatment did not. However, when F2-isoprostanes (F2-IsoPs) were measured in tissues and urine, PB showed the strongest effect and βNF had a measurable but weaker effect. The same trend was seen when an Nfr2-based transgene reporter sensitive to ROS was analyzed in the mice. This pattern had been seen earlier with F2-IsoPs both in vitro and in vivo with rats (Dostalek, M., Brooks, J. D., Hardy, K. D., Milne, G. L., Moore, M. M., Sharma, S., Morrow, J. D., and Guengerich, F. P. (2007) Mol. Pharmacol. 72, 1419–1424). One possibility for the general in vitro-in vivo discrepancy in oxidative stress found in both mice and rats is that PB treatment might attenuate protective systems. One potential candidate suggested by an mRNA microarray was nicotinamide N-methyltransferase. PB was found to elevate nicotinamide N-methyltransferase activity 3- to 4-fold in mice and rats and to attenuate levels of NAD+, NADP+, NADH, and NADPH in both species (20–40%), due to the enhanced excretion of (N-methyl)nicotinamide. PB also down-regulated glutathione peroxidase and glutathione reductase, which together constitute a key enzymatic system that uses NADPH in protecting against oxidative stress. These multiple effects on the protective systems are proposed to be more important than P450 induction in oxidative stress and emphasize the importance of studying in vivo models.

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication.

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3′ to 5′ exonuclease activities in vitro. Steady-state kinetic efficiencies (kcat/Km) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.

Metabolism of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine by Mitochondrion-targeted Cytochrome P450 2D6 IMPLICATIONS IN PARKINSON DISEASE

Background: Metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic MPP+ is critical in chemically induced Parkinson disease. Results: Mitochondrial CYP2D6 supported by adrenodoxin/adrenodoxin reductase efficiently catalyzed MPTP to MPP+. Conclusion: Mitochondria from dopaminergic neurons contain the enzymes for the metabolism of MPTP to MPP+. Significance: This is a new pathway for the metabolism of MPTP to toxic MPP+ within the dopaminergic neurons. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxic side product formed in the chemical synthesis of desmethylprodine opioid analgesic, which induces Parkinson disease. Monoamine oxidase B, present in the mitochondrial outer membrane of glial cells, catalyzes the oxidation of MPTP to the toxic 1-methyl-4-phenylpyridinium ion (MPP+), which then targets the dopaminergic neurons causing neuronal death. Here, we demonstrate that mitochondrion-targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the metabolism of MPTP to MPP+, as shown with purified enzymes and also in cells expressing mitochondrial CYP2D6. Neuro-2A cells stably expressing predominantly mitochondrion-targeted CYP2D6 were more sensitive to MPTP-mediated mitochondrial respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Mitochondrial CYP2D6 expressing Neuro-2A cells produced higher levels of reactive oxygen species and showed abnormal mitochondrial structures. MPTP treatment also induced mitochondrial translocation of an autophagic marker, Parkin, and a mitochondrial fission marker, Drp1, in differentiated neurons expressing mitochondrial CYP2D6. MPTP-mediated toxicity in primary dopaminergic neurons was attenuated by CYP2D6 inhibitor, quinidine, and also partly by monoamine oxidase B inhibitors deprenyl and pargyline. These studies show for the first time that dopaminergic neurons expressing mitochondrial CYP2D6 are fully capable of activating the pro-neurotoxin MPTP and inducing neuronal damage, which is effectively prevented by the CYP2D6 inhibitor quinidine.

Human Cytochrome P450 2E1 Mutations That Alter Mitochondrial Targeting Efficiency and Susceptibility to Ethanol-induced Toxicity in Cellular Models

Background: Induced expression of CYP2E1 is known to enhance alcohol liver toxicity. Results: Novel mutations W23R/W30R and L32N in human CYP2E1 alter mitochondrial and microsomal targeting efficiency. Conclusion: Human variants with altered targeting modulate susceptibility of cells to alcohol. Significance: Carriers of the novel W23R/W30R mutation in CYP2E1 are likely to be more susceptible to alcohol toxicity. Human polymorphisms in the 5′-upstream regulatory regions and also protein coding regions of cytochrome P450 2E1 (CYP2E1) are known to be associated with several diseases, including cancer and alcohol liver toxicity. In this study, we report novel mutations in the N-terminal protein targeting regions of CYP2E1 that markedly affect subcellular localization of the protein. Variant W23R/W30R protein (termed W23/30R) is preferentially targeted to mitochondria but very poorly to the endoplasmic reticulum, whereas the L32N protein is preferentially targeted to the endoplasmic reticulum and poorly to mitochondria. These results explain the physiological significance of bimodal CYP targeting to the endoplasmic reticulum and mitochondria previously described. COS-7 cells and HepG2 cells stably expressing W23/30R mutations showed markedly increased alcohol toxicity in terms of increased production of reactive oxygen species, respiratory dysfunction, and loss of cytochrome c oxidase subunits and activity. Stable cells expressing the L32N variant, on the other hand, were relatively less responsive to alcohol-induced toxicity and mitochondrial dysfunction. These results further support our previous data, based on mutational studies involving altered targeting, indicating that mitochondria-targeted CYP2E1 plays an important role in alcohol liver toxicity. The results also provide an interesting new link to genetic variations affecting subcellular distribution of CYP2E1 with alcohol-induced toxicity.