Expression, purification and bioactivity of human augmenter of liver regeneration.
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AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. METHODS: hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS: The product of PCR from plasmid pGEM-T-hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P < 0.01). CONCLUSION: Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application. (+info)
Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei.
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Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle. (+info)
Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria.
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A number of cytosines are altered to be recognized as uridines in transcripts of the nad3 locus in mitochondria of the higher plant Oenothera. Such nucleotide modifications can be found at 16 different sites within the nad3 coding region. Most of these alterations in the mRNA sequence change codon identities to specify amino acids better conserved in evolution. Individual cDNA clones differ in their degree of editing at five nucleotide positions, three of which are silent, while two lead to codon alterations specifying different amino acids. None of the cDNA clones analysed is maximally edited at all possible sites, suggesting slow processing or lowered stringency of editing at these nucleotides. Differentially edited transcripts could be editing intermediates or could code for differing polypeptides. Two edited nucleotides in an open reading frame located upstream of nad3 change two amino acids in the deduced polypeptide. Part of the well-conserved ribosomal protein gene rps12 also encoded downstream of nad3 in other plants, is lost in Oenothera mitochondria by recombination events. The functional rps12 protein must be imported from the cytoplasm since the deleted sequences of this gene are not found in the Oenothera mitochondrial genome. The pseudogene sequence is not edited at any nucleotide position. (+info)
Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland.
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1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential ultracentrifugation. 2. Neurosecretory vesicles were isolated by sucrose-gradient ultracentrifugation and membranes were obtained after hypo-osmotic lysis of the particles. 3. A method is described for the isolation of a preparation of purified neuronal plasma membranes by using a fraction enriched in nerve endings as a starting material. 4. The purity of the subcellular fractions was estimated by enzyme assays and by examination with the electron microscope. 5. On the basis of the results it was estimated that neuronal plasma membranes constitute more than 30% of the protein of the nerve endings and neurosecretory vesicles more than 45% of the total amount of protein in the homogenate. 6. The proteins of membranes of neurosecretory vesicles and of plasma membranes were solubilized by means of sodium dodecyl sulphate. Polyacrylamide-gel electrophoresis of such preparations showed that both membranes contained a large number of proteins, including three glycoproteins. (+info)
Effect of a single oral dose of methanol, ethanol and propan-2-ol on the hepatic microsomal metabolism of foreign compounds in the rat.
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Methanol and ethanol administered to rats as a single oral dose increased aniline hydroxylation by the hepatic microsomal fraction by a maximum of 169 and 66% respectively, whereas aminopyrine demethylation was inhibited by 51 and 61%. The concentration of microsomal cytochrome P-450, and the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were unchanged. Propan-2-ol, administered as a single oral dose, increased microsomal aniline hydroxylation by 165% and increased aminopyrine demethylation by 83%. The concentration of cytochrome P-450 was unchanged whereas NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were both increased by 38%. Methanol, ethanol and propan-2-ol administration resulted in a decreased type I spectral change but had no effect on the reverse type I spectral change. Methanol administration decreased the type II spectral change whereas ethanol and propan-2-ol had no effect. Cycloheximide blocked the increases in aniline hydroxylation and aminopyrine demethylation but could not completely prevent the decreases in aminopyrine demethylation. The increases in aniline hydroxylation were due to an increase in V, but Km was unchanged. The ability of acetone to enhance and compound SKF 525A to inhibit microsomal aniline hydroxylation was decreased by the administration of all three alcohols. The decrease in the metabolism of aminopyrine may result from a decrease in the binding to the type I site with a consequent failure of aminopyrine to stimulate the reduction of cytochrome P-450. Methanol administration may lead to an increase in aniline hydroxylation because of a failure of aniline to inhibit cytochrome P-450 reduction. (+info)
Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes.
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The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin binding to the solubilized receptor has been developed. Both the solubilized thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes have effectively identical nonlinear Scatchard plots and negatively sloped Hill plots, i.e. both preparations have receptors which appear to exhibit a similar negatively cooperative relationship. Although the pH optimum of thyrotropin binding to the solubilized receptor is the same as that of the thyroid plasma membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor is slightly different in its outline. Thyrotropin binding to the solubilized receptor is less sensitive to salt inhibition than is binding to the thyroid plasma membrane receptor; however, optimal binding remains at 0 degrees. The relative affinities of thyrotropin and two glycoprotein hormones which can be considered structural analogs, luteinizing hormone and human chorionic gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but 100:25:40 toward the solubilized receptors. The solubilized receptor preparation is heterogeneous in size in that it has binding components with molecular weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion converts all three higher molecular weight components to the 15,000 to 30,000 molecular weight species, and the 15,000 to 30,000 molecular weight receptor component has all of the binding properties of the solubilized receptor preparation before tryptic digestion including an identical nonlinear Scatchard plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to 30,000 molecular weight receptor released by tryptic digestion of bovine thyroid plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture. The tryptic fragment of the solubilized receptor or preparations has been purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose columns. The binding activity is lost when the solubilized thyrotropin receptor preparation is exposed to beads of neuraminidase-Sepharose or conconavalin A-Sepharose. (+info)
Enzyme source effects on CYP2C9 kinetics and inhibition.
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When choosing a recombinant cytochrome P450 (P450) enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. Although literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes, Baculosomes, RECO system, and in-house purified, reconstituted enzyme) to determine whether the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher K(m) values, reduced substrate affinity, and lower calculated intrinsic clearance values compared with baculovirus microsomal preparations. Six- to 25-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. In addition, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of 12 inhibitors, decreased inhibition potency was observed across 11 of those inhibitors in the RECO purified, reconstituted enzyme compared with the Supersomes baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of an enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9. (+info)
Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.
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Mutants of Saccharomyces cereviaiae showing defects in cytochrome oxidase, coenzyme QH2-cytochrome c reductase, and rutamycin-sensitive ATPase are described. The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 2:2 segregation of tetrads. Genetic analysis indicate the coenzyme QH2-cytochrome c reductase and cytochrome oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F1 ATPase, while the third synthesizes F1 but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex. (+info)