Purified Cytochrome P-450 Research Articles

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

Anthochlor pigments (chalcones and aurones) play an important role in yellow flower colourization, the formation of UV-honey guides and show numerous health benefits. The B-ring hydroxylation of chalcones is performed by membrane bound cytochrome P450 enzymes. It was assumed that usual flavonoid 3′-hydroxlases (F3′Hs) are responsible for the 3,4- dihydroxy pattern of chalcones, however, we previously showed that a specialized F3′H, namely chalcone 3-hydroxylase (CH3H), is necessary for the hydroxylation of chalcones. In this study, a sequence encoding membrane bound CH3H from Dahlia variabilis was recombinantly expressed in yeast and a purification procedure was developed. The optimized purification procedure led to an overall recovery of 30% recombinant DvCH3H with a purity of more than 84%. The enzyme was biochemically characterized with regard to its kinetic parameters on various substrates, including racemic naringenin, as well as its enantiomers (2S)-, and (2R)-naringenin, apigenin and kaempferol. We report for the first time the characterization of a purified Cytochrome P450 enzyme from the flavonoid biosynthesis pathway, including the transmembrane helix. Further, we show for the first time that recombinant DvCH3H displays a higher affinity for (2R)-naringenin than for (2S)-naringenin, although (2R)-flavanones are not naturally formed by chalcone isomerase.

Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism.We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed.The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT.A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.

Non‐Additive Effects of Changing the Composition of the Cytochrome P450 Ensemble in Human Liver Microsomes: the Focus on CYP2E1 and Alcohol‐Drug Interactions

Recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a straight correlation between the composition of human cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of physical interactions between multiple P450 species co‐localized in the microsomal membrane. Due to complex effects of these interactions any change in the content of a particular P450 enzyme may affect drug metabolism in a complex, hard‐to‐predict manner. Of particular practical importance are the functional effects of the changes in the composition of P450 ensemble induced by alcohol consumption. Although the multi‐fold increase in the content of cytochrome P450 2E1 (CYP2E1) in liver observed in both alcoholics and moderate alcohol consumers is well documented, the role of CYP2E1 in the instances of alcohol‐drug interactions is commonly considered insignificant due to a minor role of this enzyme in drug metabolism. However, the involvement of CYP2E1 in alcohol‐drug interactions may stretch far beyond its immediate effects on the metabolism of its substrates.The aim of this study is to probe the effect of increasing concentrations of CYP2E1 in HLM on the metabolism of drug substrates of other P450 species. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into HLM that allows creating the microsomal preparations with altered composition of the P450 ensemble. Incorporation of CYP2E1 into HLM results in an increase in the rate of CYP2E1‐dependent metabolism of chlorzoxazone, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Studies with a series of substrates of drug‐metabolizing P450 species showed that CYP2E1 incorporation also results in pronounced alterations in the metabolism of substrates of several other P450 enzymes. The most important effect is observed on CYP2C19. The changes in the effective KM and Vmax values with such probe substrates as 7‐etoxy‐4‐cyanocoumarin (CEC, the substrate of CYP2C19 and CYP1A2), 7‐dimethylamino‐4‐trifluoromethylcoumarin (coumarin‐152, the substrate of CYP2B6, CYP2C19 and CYP2C9) and N‐butyl 7‐methoxy‐4‐(aminomethyl)‐coumarin (BMAMC, the substrate of CYP1A2, CYP2C19 and CYP2D6) reveal a substantial decrease in the involvement of CYP2C19 in their metabolism. Furthermore, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5. These observations suggest that ethanol‐dependent induction CYP2E1 may be involved in pronounced pharmacokinetic interactions of alcohol with such drug substrates of CYP2C19 and CYP3A enzymes as benzodiazepines (diazepam), tricyclic antidepressants (amitriptylline, imipramine) and anticonvulsants (phenytoin, carbamazepine).Support or Funding InformationThis research was supported by grant R21‐AA024548 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Towards a System Approach to the Human Cytochrome P450 Ensemble: A New Strategy for Studying the Network of P450‐P450 Interactions in Human Liver Microsomes

The composition of the human cytochrome P450 ensemble is commonly recognized as a key factor determining the interindividual variability in drug metabolism. However, recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a direct correlation between the composition of the cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of interactions between multiple cytochrome P450 species co‐localized in the microsomal membrane. A change in the content of any particular P450 enzyme alters the landscape of protein‐protein interactions and thus affects drug metabolism in a complex, hard‐to‐predict manner. Therefore, a systematic exploration of the network of P450‐P450 interactions and identification of their functional consequences represents an issue of emerging importance for revealing the interrelationships between the profile of P450 expression and the profile of drug metabolism. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into the microsomes. Here we demonstrate high potential of this approach for creating preparations of HLM with altered composition of the P450 ensemble. An important characteristic of this strategy is its adaptability for incorporating genetically or chemically modified proteins. This includes the incorporation of proteins bearing fluorescent probes and light‐activating crosslinkers. Therefore, this strategy may be used for monitoring protein‐protein interactions in HLM with a variety of biophysical techniques, as well as for searching the partners of P450 protein‐protein interactions with the technique of molecular fishing.Here we report the use of our new approach for studying the effect of enriching the HLM with ethanol‐inducible CYP2E1, one of the the most populated P450 species in human liver. Incorporation of CYP2E1 into HLM results in an increase in the rate of metabolism of CYP2E1‐specific substrates, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Our studies with a series of substrates of drug‐metabolizing P450s showed that CYP2E1 incorporation results in pronounced alterations in the metabolism of substrates of several other P450 species present in HLM. In particular, we observed over 2‐fold increase in the rate of metabolism and a substantial decrease in the apparent KM observed with 3‐cyano‐7‐etoxycoumarin (CEC), the substrate of CYP1A2 and CYP2C19. This finding may indicate that the increase in CYP2E1 concentration results in redirecting the main route of CEC metabolism from CYP2C19 to CYP1A2, which is characterized with lower KM and higher turnover numbers with this substrate. Similarly, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax, observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5.Support or Funding InformationThis research was supported by grant R01‐GM114369 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic characteristics of Tanshinone I in human liver microsomes and S9 subcellular fractions

Tanshinone I (TSI) is a lipophilic diterpene in Salvia miltiorrhiza with versatile pharmacological activities. However, metabolic pathway of TSI in human is unknown.In this study, we determined major metabolites of TSI using a preparation of human liver microsomes (HLMs) by HPLC-UV and Q-Trap mass spectrometer. A total of 6 metabolites were detected, which indicated the presence of hydroxylation, reduction as well as glucuronidation.Selective chemical inhibition and purified cytochrome P450 (CYP450) isoform screening experiments revealed that CYP2A6 was primarily responsible for TSI Phase I metabolism. Part of generated hydroxylated TSI was glucuronidated via several glucuronosyltransferase (UGT) isoforms including UGT1A1, UGT1A3, UGT1A7, UGT1A9, as well as extrahepatic expressed isoforms UGT1A8 and UGT1A10. TSI could be reduced to a relatively unstable hydroquinone intermediate by NAD(P)H: quinone oxidoreductase 1 (NQO1), and then immediately conjugated with glucuronic acid by a panel of UGTs, especially UGT1A9, UGT1A1 and UGT1A8. Additionally, NQO1 could also reduce hydroxylated TSI to a hydroquinone intermediate, which was immediately glucuronidated by UGT1A1.The study demonstrated that hydroxylation, reduction as well as glucuronidation were the major pathways for TSI biotransformation, and six metabolites generated by CYPs, NQO1 and UGTs were found in HLMs and S9 subcellular fractions.

Cytochrome P450 fromPhotobacterium profundumSS9, a Piezophilic Bacterium, Exhibits a Tightened Control of Water Access to the Active Site

We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further toward the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoylglycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoylglycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around -100 to -200 mL/mol and P(1/2) of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium toward the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.

Purification of CYP2B‐like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Mechanism of inhibition of purified leaping mullet (liza saliens) NADPH‐cytochrome P450 reductase by toxic metals: Aluminum and thallium

Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.

耐熱性シトクロムＰ４５０のポリエチレンオキシド中における酸化還元応答に及ぼす支持電解質の効果―バイオカソード用触媒としての利用を目指して

Cytochrome P450s are heme-thiolate enzymes found in organisms from all domains of life. They are involved in a wide variety of biological oxidation reactions. We reported overexpression and purification of cytochrome P450 from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st), previously. In this study, P450st has been modified with poly (ethylene oxide) (PEO) to dissolve in PEO200. The electron transfer reaction between PEO2000 modified P450st (PEO2000-P450st) and a plastic formed carbon electrode in PEO200 containing 0.5 M KCl was investigated. A clear redox couple was observed at -114 mV vs. Ag. This was assigned to the FeII/FeIII. In PEO200 containing 0.5 M KBr, the redox potential for PEO2000-P450st was 52 mV vs. Ag wire. The results show that anions are important to control the redox potential for cytochrome P450.

Purification of recombinant wheat cytochrome P450 monooxygenase expressed in yeast and its properties

To investigate the properties of wheat cytochrome P450 and the characteristics of herbicide metabolism by cytochrome P450 in vitro, deeply understand the mechanisms of herbicide selectivity, recombinant wheat cytochrome P450 monooxygenase (CYP71Cv1) heterologously expressed in yeast was purified by DE-52 cellulose chromatography and fast protein liquid chromatography (FPLC) with Mono-Q column. The degree of purification was 1366-fold. The specific activity of purified cytochrome P450 reached to 512 nmol min −1 mg −1 protein with herbicide chlorsulfuron as substrate. The purified cytochrome P450 exhibited one band in sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, and the molecular mass was 52.5 kDa. Kinetic parameter was determined in vitro. The K m values for chlorsulfuron and triasulfuron were 57 (±15) and 38 (±16) μM, respectively; and V max for chlorsulfuron and triasulfuron were 4.1 (±0.7) and 2.7 (±0.5) nmol min −1 mg −1 protein in vitro, respectively.

ROLE OF CYTOCHROMEB5IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.

Biosynthesis of β-Substituted Furan Skeleton in the Lower Furanoterpenoids: A Model Study

Furanoterpenes are widely distributed in the plant kingdom. In this study we have carried out enzymatic synthesis of simple furan compounds from the molecules containing an α-isopropylidene ketone unit and the role of cytochrome P450 in this biotransformation has been conclusively established. Eight model compounds (acyclic, monocyclic, and bicyclic, 1–8), having an α-isopropylidene ketone unit, were synthesized and incubated with PB-induced rat liver microsomes in the presence of NADPH and O2. GC–MS and NMR analyses of the product(s) indicated the formation of corresponding furano derivatives (11–18). Cytochrome P450 inhibitors, metyrapone, SKF-525, and carbon monoxide, inhibited the formation of furan (8) to 76, 62, and 97%, respectively. Incubation of dehydrofukinone (7), a naturally occurring terpene, with purified cytochrome P450, NADPH–cytochrome P450 reductase, and dilaurylphosphatidylcholine in the presence of NADPH and O2 resulted in the formation of 10 and furanodehydrofukinone (19). Based on these observations, we propose one of the probable biosynthetic routes for lower furanoterpenoids in higher plants.

Purification of cytochromes P-450 scc and P-450 17α by steroid-binding affinity column chromatography

The preparation, testing and use of a variety of cholesterol-, deoxycorticosterone (DOC)- and pregnenolone-binding 1,6-diaminohexyl (EAH)-Sepharose 4B supports for affinity column chromatography of cytochromes P-450 scc and P-450 17α from bovine adrenal and pig testis are described. EAH-Sepharose 4B has free amino groups at the end of a 10-atom spacer arm. Hydroxyl groups of cholesterol (3β), deoxycorticosterone (21β) and pregnenolone (3β) are linked to succinic anhydride in pyridine through an ester linkage. These coupling ligands of hemisuccinate were synthesized by a general procedure. Free amino groups of EAH-Sepharose 4B were used to couple ligands, containing carboxyl groups, by the carbodiimide coupling method. Both the purified cytochromes P-450 scc and P-450 17α were found to be homogeneous and estimated to have a molecular weight of 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectra with peaks at 450 and 448 nm exhibit the absorption spectra of typical cytochromes P-450 scc and P-450 17α, respectively. Cytochromes P-450 scc and P-450 17α were determined to have isoelectric points of 8.0 and 6.5 in isoelectric focusing on a pH gradient gel. Cytochrome P-450s can be purified between 425- and 1000-fold from the crude extracts.

Inactivation of cytochrome P450 2B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables.

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time- and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K(I)) was 5.8 microM, and the maximal rate constant for inactivation was 0.66 min(-)(1) at 23 degrees C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 +/- 0. 16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

A Simplified Procedure for Purification of Cytochrome P-450 by Preparative Ampholine Gel for Isoelectric Focusing

ABSTRACT The purification process for cytochrome P-450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P-450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.

A two-step purification of cytochrome P-450 from adult pig testis by pregnenolone affinity column chromatography.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P-450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A purification of 755x was achieved with a yield of 3.09%.

Purification, reconstitution, and inhibition of cytochrome P-450 sterol delta22-desaturase from the pathogenic fungus Candida glabrata.

Sterol delta22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14alpha-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol delta22-desaturase activity in a reconstituted system with NADPH-cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 microM and a Vmax of 0. 59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol delta22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol delta22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing.

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.