Synthesis and characterization of 3H-labelled tetrahydrobiopterin.
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ABSTRACT:
SUBMITTER: Werner ER
PROVIDER: S-EPMC1137470 | biostudies-other | 1994 Nov
REPOSITORIES: biostudies-other
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Project description:We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH₄) in the lipid-producing fungus Mortierella alpina. The BH₄ cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H₂-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H₂-NTP to 6-pyruvoyltetrahydropterin (PPH₄), and sepiapterin reductase (SR) for the conversion of PPH₄ to BH₄, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH₄ in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH₄ and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH₄ biosynthesis pathway in a fungus.
| S-EPMC4811656 | biostudies-literature
Project description:Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.
| S-EPMC1135094 | biostudies-other
Project description: Not available
| S-EPMC10915669 | biostudies-literature
Project description:The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.
| S-EPMC1162317 | biostudies-other
Project description:Binding of 3H-labelled platelet-activating factor ([3H]PAF) to guinea-pig tracheal epithelial cells was time-dependent, reversible and saturable. Scatchard analysis of the saturation-binding data indicated that [3H]PAF bound to one class of specific binding sites with high affinity (KD = 4.3 +/- 0.03 nM; Bmax. = 0.172 +/- 0.02 fmol/10(5) cells; n = 3). Unlabelled PAF competitively and selectively inhibited the specific binding of [3H]PAF with 50% inhibition at 4.8 +/- 0.07 nM (n = 3). SR 27417, the first member of a newly developed PAF antagonist series, competitively displaced [3H]PAF from its binding sites on guinea-pig tracheal epithelial cells with a Ki of 100 +/- 3 pM (n = 3). Studies carried out in parallel demonstrated that SR 27417 was 40 times more potent than C16-PAF itself and more than 100-fold as active as the best synthetic PAF-receptor antagonist yet described. [3H]SR 27417 displayed high-affinity, specific, reversible as well as saturable binding to a single class of binding sites on tracheal epithelial cells (KD = 94 +/- 7 pM; Bmax. = 0.181 +/- 0.04 fmol/10(5) cells; n = 3). C16-PAF, lyso-PAF, enantio-PAF, SR 27417 and other PAF-receptor antagonists had Ki values which were nearly identical for both [3H]PAF and [3H]SR 27417, demonstrating that in guinea-pig tracheal epithelial cells they have the same binding sites. In conclusion, these data suggest that tracheal epithelial cells contain PAF-specific receptors and indicate that SR 27417 is an extremely potent PAF-receptor antagonist, as well as being a suitable radioligand for labelling PAF receptors on intact cells.
| S-EPMC1132716 | biostudies-other
Project description:The kinetics of incorporation of leucine, galactose and mannose into intracellular and secreted myeloma protein, MOPC 21 IgG(1) and MOPC 46 kappa-type light chain, by cell suspensions of two myeloma plasma-cell tumours, MOPC 21 and MOPC 46, were similar. Radioactive galactose was incorporated to over 90% into galactose residues of intracellular and secreted protein, mannose to over 90% into glucosamine and mannose residues of intracellular protein and to over 90% into glucosamine, mannose and fucose residues of secreted protein, but not into galactose residues. The results show that specific residues in the carbohydrate portion of myeloma proteins can be labelled by specific radioactive monosaccharides, and suggest that fucose residues are added, while myeloma protein is in its final stage of secretion from the plasma cell. The kinetics of incorporation indicate at least three sequential precursor-product relationships between different intracellular forms and the secreted form of myeloma protein.
| S-EPMC1179464 | biostudies-other
Project description:BackgroundTetrahydrobiopterin (BH4) deficiency is a rare cause of hyperphenylalaninemia (HPA). The incidence of this condition varies based on region and ethnicity. In the early stages, patients typically do not exhibit any symptoms, and HPA is identified only through newborn screening for diseases. It is important to distinguish BH4 deficiency from phenylketonuria (PKU, MIM # 261600). Timely diagnosis and treatment of BH4 deficiency are crucial for the prognosis of patients.Case presentationWe present two rare cases of Chinese Tibetan children with BH4D, diagnosed through biochemical tests and genetic sequencing. Case 1 is a male infant, 2 months old, with a newborn screening (NBS) Phe level of 1212 μmol/L (reference range <120 μmol). The biopterin(B) level was 0.19 mmol/molCr (reference range: 0.42-1.92 mmol/molCr), with a B% of 5.67% (reference range: 19.8%-50.3%). Gene sequencing revealed a homozygous missense variant [NM_000317.3 (PTS): c.259C > T (p.Pro87Ser), rs104894276, ClinVar variation ID: 480]. The patient was treated with a Phe-reduced diet and oral sapropterin, madopar and is currently 3 years and 4 months old, showing mild global developmental delay. Case 2 is a 40-day-old female infant with a Phe level of 2442.11 μmol/L and dihydropteridine reductase (DHPR) activity of 0.84 nmol/(min. 5 mm disc) (reference range: 1.02-3.35 nmol/min.5 mm disc. Gene sequencing revealed a compound heterozygous genotype [NM_000320.3(QDPR): c.68G > A (p.Gly23Asp), rs104893863, ClinVar Variation ID: 490] and [NM_000320.3(QDPR) c.419C > A (p. Ala140Asp), ClinVar ID: 2444501]. The patient was treated with a Phe-reduced diet and oral madopar, 5-hydroxytryptophan. At the age of 1 year, she exhibited severe global developmental delay with seizures.ConclusionWe identified and treated two cases of BH4D in Tibetan populations in China, marking the first confirmed instances. Our report emphasizes the significance of conducting differential diagnosis tests for BH4D.
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Project description:Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.
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Project description:Human neutrophil elastase (HNE) is a potent serine protease belonging to the chymotrypsin family and is involved in a variety of pathologies affecting the respiratory system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here the synthesis of new thiazol-2-(3H)-ones as an elaboration of potent HNE inhibitors with an isoxazol-5-(2H)-one scaffold that we recently identified. Two-dimensional NMR spectroscopic techniques and tandem mass spectrometry allowed us to correctly assign the structure of the final compounds arising from both tautomers of the thiazol-2-(3H)-one nucleus (N-3 of the thiazol-2-(3H)-one and 3-OH of the thiazole). All new compounds were tested as HNE inhibitors, and no activity was found at the highest concentration used (40 µM), demonstrating that the thiazol-2-(3H)-one is not a good scaffold for HNE inhibitors. Molecular modelling experiments indicate that the low-energy pose might limit the nucleophilic attack on the endocyclic carbonyl group of the thiazolone-based compounds by HNE catalytic Ser195, in contrast to isoxazol-5-(2H)-one analogues.
| S-EPMC5718994 | biostudies-literature
Project description:Nitric oxide (NO), a key regulator of cardiovascular function, is synthesized from L-arginine and oxygen by the enzyme nitric oxide synthase (NOS). This reaction requires tetrahydrobiopterin (BH4) as a cofactor. BH4 is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GTPCH) and recycled from 7,8-dihydrobiopterin (BH2) by dihydrofolate reductase. Under conditions of low BH4 bioavailability relative to NOS or BH2, oxygen activation is "uncoupled" from L-arginine oxidation, and NOS produces superoxide (O (2) (-) ) instead of NO. NOS-derived superoxide reacts with NO to produce peroxynitrite (ONOO(-)), a highly reactive anion that rapidly oxidizes BH4 and propagates NOS uncoupling. BH4 depletion and NOS uncoupling contribute to overload-induced heart failure, hypertension, ischemia/reperfusion injury, and atrial fibrillation. L-arginine depletion, methylarginine accumulation, and S-glutathionylation of NOS also promote uncoupling. Recoupling NOS is a promising approach to treating myocardial and vascular dysfunction associated with heart failure.
| S-EPMC3406312 | biostudies-literature