Newly biosynthesized cytochrome P-450 associated with the golgi apparatus from livers of rats induced with phenobarbital.
Ontology highlight
ABSTRACT:
SUBMITTER: Cooper MB
PROVIDER: S-EPMC1162803 | biostudies-other | 1981 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.
| S-EPMC1152009 | biostudies-other
Project description:Cytochrome P450s (CYPs) are phase-I metabolic enzymes playing important roles in drug metabolism, dietary chemicals and endogenous molecules. Age is a key factor influencing P450s expression. Thus, age-related changes of CYP 1-4 families and bile acid homeostasis-related CYPs, the corresponding nuclear receptors and a few phase-II genes were examined. Livers from male Sprague-Dawley rats at fetus (-2 d), neonates (1, 7, and 14 d), weanling (21 d), puberty (28 and 35 d), adulthood (60 and 180 d), and aging (540 and 800 d) were collected and subjected to qPCR analysis. Liver proteins from 14, 28, 60, 180, 540 and 800 days of age were also extracted for selected protein analysis by western blot. In general, there were three patterns of their expression: Some of the drug-metabolizing enzymes and related nuclear receptors were low in fetal and neonatal stage, increased with liver maturation and decreased quickly at aging (AhR, Cyp1a1, Cyp2b1, Cyp2b2, Cyp3a1, Cyp3a2, Ugt1a2); the majority of P450s (Cyp1a2, Cyp2c6, Cyp2c11, Cyp2d2, Cyp2e1, CAR, PXR, FXR, Cyp7a1, Cyp7b1. Cyp8b1, Cyp27a1, Ugt1a1, Sult1a1, Sult1a2) maintained relatively high levels throughout the adulthood, and decreased at 800 days of age; and some had an early peak between 7 and 14 days (CAR, PXR, PPARα, Cyp4a1, Ugt1a2). The protein expression of CYP1A2, CYP2B1, CYP2E1, CYP3A1, CYP4A1, and CYP7A1 corresponded the trend of mRNA changes. In summary, this study characterized three expression patterns of 16 CYPs, five nuclear receptors, and four phase-II genes during development and aging in rat liver, adding to our understanding of age-related CYP expression changes and age-related disorders.
| S-EPMC6681801 | biostudies-literature
Project description:The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.
| S-EPMC1144199 | biostudies-other
Project description:Autophagy has dual effects in human diseases: appropriate autophagy may protect cells from stress, while excessive autophagy may cause cell death. Additionally, close interactions exist between autophagy and the Golgi. This review outlines recent advances regarding the role of the Golgi apparatus in autophagy. The signaling processes of autophagy are dependent on the normal function of the Golgi. Specifically, (i) autophagy-related protein 9 is mainly located in the Golgi and forms new autophagosomes in response to stressors; (ii) Golgi fragmentation is induced by Golgi-related proteins and accompanied with autophagy induction; and (iii) the endoplasmic reticulum-Golgi intermediate compartment and the reticular trans-Golgi network play essential roles in autophagosome formation to provide a template for lipidation of microtubule-associated protein 1A/1B-light chain 3 and induce further ubiquitination. Golgi-related proteins regulate formation of autophagosomes, and disrupted formation of autophagy can influence Golgi function. Notably, aberrant autophagy has been demonstrated to be implicated in neurological diseases. Thus, targeted therapies aimed at protecting the Golgi or regulating Golgi proteins might prevent or ameliorate autophagy-related neurological diseases. Further studies are needed to investigate the potential application of Golgi therapy in autophagy-based neurological diseases.
| S-EPMC7509445 | biostudies-literature
Project description:Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.
| S-EPMC1152366 | biostudies-other
Project description:Golgi apparatus (GA) oxidative stress induced by in situ reactive oxygen species (ROS) could severely damage the morphology and function of GA, which may open up an avenue for effective photodynamic therapy (PDT). However, due to the lack of effective design strategy, photosensitizers (PSs) with specific GA targeting ability are in high demand and yet quite challenging. Herein, we report an aggregation-induced emission luminogen (AIEgen) based PS (TPE-PyT-CPS) that can effectively target the GA via caveolin/raft mediated endocytosis with a Pearson correlation coefficient up to 0.98. Additionally, the introduction of pyrene into TPE-PyT-CPS can reduce the energy gap between the lowest singlet state (S1) and the lowest triplet state (T1) (ΔEST) and exhibits enhanced singlet oxygen generation capability. GA fragmentation and cleavage of GA proteins (p115/GM130) are observed upon light irradiation. Meanwhile, the apoptotic pathway is activated through a crosstalk between GA oxidative stress and mitochondria in HeLa cells. More importantly, GA targeting TPE-T-CPS show better PDT effect than its non-GA-targeting counterpart TPE-PyT-PS, even though they possess very close ROS generation rate. This work provides a strategy for the development of PSs with specific GA targeting ability, which is of great importance for precise and effective PDT.
| S-EPMC9023483 | 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: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:LC3 is a protein that can associate with autophagosomes, autolysosomes, and phagosomes. Here, we show that LC3 can also redistribute toward the damaged Golgi apparatus where it clusters with SQSTM1/p62 and lysosomes. This organelle-specific relocation, which did not involve the generation of double-membraned autophagosomes, could be observed after Golgi damage was induced by various strategies, namely (i) laser-induced localized cellular damage, (ii) local expression of peroxidase and exposure to peroxide and diaminobenzidine, (iii) treatment with the Golgi-tropic photosensitizer redaporfin and light, (iv) or exposure to the Golgi-tropic anticancer peptidomimetic LTX-401. Mechanistic exploration led to the conclusion that both reactive oxygen species-dependent and -independent Golgi damage induces a similar phenotype that depended on ATG5 yet did not depend on phosphatidylinositol-3-kinase catalytic subunit type 3 and Beclin-1. Interestingly, knockout of ATG5 sensitized cells to Golgi damage-induced cell death, suggesting that the pathway culminating in the relocation of LC3 to the damaged Golgi may have a cytoprotective function.
| S-EPMC6748261 | biostudies-literature
Project description:The Golgi apparatus is an essential organelle of the secretory pathway in eukaryotic cells. It processes secretory and transmembrane proteins and orchestrates their transport to other endomembrane compartments or the plasma membrane. The Golgi apparatus thereby shapes the cell surface, controlling cell polarity, cell-cell communication, and immune signaling. The cytosolic face of the Golgi hosts and regulates signaling cascades, impacting most notably the DNA damage response and mitosis. These essential functions strongly depend on Golgi protein homeostasis and Golgi integrity. Golgi fragmentation and consequent malfunction is associated with neurodegenerative diseases and certain cancer types. Recent studies provide first insight into the critical role of ubiquitin signaling in maintaining Golgi integrity and in Golgi protein quality control. Similar to well described pathways at the endoplasmic reticulum, ubiquitin-dependent degradation of non-native proteins prevents the accumulation of toxic protein aggregates at the Golgi. Moreover, ubiquitination regulates Golgi structural rearrangements in response to cellular stress. Advances in elucidating ubiquitination and degradation events at the Golgi are starting to paint a picture of the molecular machinery underlying Golgi (protein) homeostasis.
| S-EPMC10357820 | biostudies-literature