Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.
Ontology highlight
ABSTRACT:
SUBMITTER: Paine AJ
PROVIDER: S-EPMC1161982 | biostudies-other | 1980 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The present work shows that the ability of pyridines e.g. metyrapone, to maintain the cytochrome P-450 concentration in cultured hepatocytes is not due to their ability to alter the 5-aminolaevulinate synthase and haem oxygenase activities of the hepatocytes. Since ligands such as metyrapone will prevent the cobalt-mediated loss of hepatic cytochrome P-450 in rats, the hypothesis that ligand formation is the mechanism of maintenance of the cytochrome in hepatocyte culture was tested. The observation that non-pyridine ligands will maintain the cytochrome P-450 concentration supports this hypothesis.
| S-EPMC1154299 | biostudies-other
Project description:mRNAs encoding cytochrome P-450s CYP1A2 and CYP2B1/2 have been quantified in rat hepatocytes cultured for periods up to 72 h under several different culture conditions that maintain total cytochrome P-450 content. When hepatocytes were cultured at either 37 or 30 degrees C in Williams E media, both CYP1A2 and CYP2B1/2 mRNAs declined dramatically. However, when cultured at 30 degrees C for 24 h, the decline in these mRNAs was not as great as that observed in cells grown at 37 degrees C. The addition of dimethyl sulphoxide to cells grown at 37 degrees C did not affect the rate of disappearance of the CYP1A2 or CYP2B1/2 mRNAs. These mRNAs also declined rapidly in cells grown in 'P-450 medium' i.e. RPMI 1640 medium without cyst(e)ine but supplemented with 0.1 mM-delta-aminolaevulinic acid. However, the levels of CYP2B1/2 mRNAs were maintained when hepatocytes were cultured in Williams E medium supplemented with 0.5 mM-metyrapone. These conditions did not, however, maintain the levels of CYP1A2 mRNA.
| S-EPMC1132884 | biostudies-other
Project description:Cytochrome P450 (CYP) proteins constitute a large ancient family of oxidative enzymes essential for the efficient elimination of a wide variety of clinically used drugs. Polymorphic variants of human CYP2D6 are associated with the conversion rate and efficacy of several drugs such as antidepressants. Polymorphisms of the canine orthologue CYP2D15 are of interest because these antidepressants are also used in dogs with behavioral problems and the outcome of the treatment is variable. However, the annotated CYP2D15 gene is incomplete and inaccurately assembled in CanFam3.1, hampering DNA sequence analysis of the gene in individual dogs. We elucidated the complete exon-intron structure of CYP2D15 to enable comprehensive genotyping of the gene using genomic DNA. We surveyed variations of the gene in four diverse dog breeds and identified novel polymorphisms in exon 2 in border collies. Further investigation to establish the impact of these canine CYP2D15 polymorphisms on interindividual variability in expression and function of this metabolizing enzyme is now feasible. Further knowledge of CYP pharmacogenetics will help individualize therapy and thereby increase therapeutic efficacy, especially in the use of antidepressants in veterinary behavioral medicine.
| S-EPMC7689907 | biostudies-literature
Project description:Ligands bind to an occluded orthosteric ligand-binding pocket within the nuclear receptor ligand-binding domain. Molecular simulations have revealed theoretical ligand entry/exit pathways to the orthosteric pocket; however, it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Here, using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and surface plasmon resonance analysis, we provide evidence that structurally distinct agonists bind peroxisome proliferator-activated receptor γ (PPARγ) via a two-step induced fit mechanism involving an initial fast kinetic step followed by a slow conformational change. The agonist encounter complex binding pose is suggested in crystal structures where ligands bind to a surface pore suggested as a ligand entry site in molecular simulations. Our findings suggest an activation mechanism for PPARγ whereby agonist binding occurs through an initial encounter complex followed by a transition of the ligand into the final binding pose within the orthosteric pocket, inducing a transcriptionally active conformation.
| S-EPMC8418994 | biostudies-literature
Project description:Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.
| S-EPMC2742861 | biostudies-other
Project description:BackgroundCYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of colorectal cancer (CRC). In the past decade, the relationship between CYP2C9 common polymorphisms (R144C and I359L) and CRC has been reported in various ethnic groups; however, these studies have yielded contradictory results. To investigate this inconsistency, we performed this meta-analysis.MethodsDatabases including Pubmed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.ResultsA total of 13 articles involving 9,463 cases and 11,416 controls were included. Overall, the summary odds ratio of CRC was 0.98 (95% CI: 0.89-1.06) and 0.99 (95% CI: 0.87-1.14) for CYP2C9 144C and 359L alleles, respectively. No significant results were observed using dominant or recessive genetic model for these polymorphisms. In the stratified analyses according to ethnicity and sex, no evidence of any gene-disease association was obtained.ConclusionsThis meta-analysis suggests that the CYP2C9 may not be associated with colorectal cancer development.
| S-EPMC3492323 | biostudies-literature
Project description:The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, β1-β2 loop, and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.
| S-EPMC3568706 | biostudies-literature
Project description:ObjectiveIn native articular cartilage, chondrocytes are surrounded by a thin pericellular matrix (PCM) forming chondrons. The PCM is exclusively rich in type VI collagen. The retention of the PCM has a significant influence on the metabolic activity of the chondrocytes.DesignThis study investigated the influence of 2 hydrogels (hyaluronic acid [HA] and agarose) and 2 media compositions (basal and chondrogenic) on the preservation/maintenance and acceleration of PCM formation over a 21-day time course. Different combinations of chondrocytes, chondrons, and mesenchymal stem cells (MSCs) were studied.ResultsBoth hydrogels preserved chondrons PCM from day 1 up to 21-day culture regardless of media composition. Type VI collagen immunostaining of the cultured chondrons appeared both dense and homogenous. The presence of MSCs did not influence this outcome. At day 1, type VI collagen was not present around chondrocytes alone or their co-culture with MSCs. In the HA hydrogel, type VI collagen was located within the PCM after 7 days in both mono- and co-cultures. In the agarose hydrogel, collagen VI was located within the PCM at 7 days (co-cultures) and 14 days (monocultures). In both hydrogel systems, chondrogenic media enhanced the production of key extracellular matrix components in both mono- and co-cultures in comparison to basal media (11.5% and 14% more in glycosaminoglycans and type II collagen for chondrocytes samples at day 21 culture samples, respectively). However, the media types did not enhance type VI collagen synthesis.ConclusionAltogether, a 3D chondrogenic hydrogel environment is the primary condition for maintenance and acceleration of PCM formation.
| S-EPMC8804781 | biostudies-literature
Project description:Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).
| S-EPMC1132036 | biostudies-other
Project description:We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
| S-EPMC2774989 | biostudies-literature