In vitro synthesis of glutathione peroxidase from selenite. Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase.     

Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [⁷⁵Se]selenocysteine and [⁷⁵Se]selenite as substrates. [⁷⁵Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [⁷⁵Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [³⁵S]cysteme, the most highly purified ⁷⁵Se-fractions were > 100-fold purified relative to ³⁵S. These fractions contained < 0.7% of the [³⁵S]cysteine originally present in the total tRNA. When [³⁵Se]selenocysteyl tRNA was purified from a mixture of ¹⁴C-labeled amino acids, over 97% of the [¹⁴C]aminoacyl tRNA was removed. The [⁷⁵Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [⁷⁵Se]selenocysteine, reaction with hydroxylamine and recovery of [⁷⁵Se]selenocysteyl hydroxamic acid and release of ⁷⁵Se by ribonuclease. The specificity of [⁷⁵Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.     

In vitro synthesis of glutathione peroxidase from selenite Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [⁷⁵Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [⁷⁵Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [³H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and ⁷⁵Se was incorporated from [⁷⁵Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the ⁷⁵Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [⁷⁵Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase     

Blood gas analysis: effect of air bubbles in syringe and delay in estimation.

Two common sources of error in blood pH and blood gas analysis were studied. The effect of delay in estimation was studied in 10 volunteers and 40 patients. Syringes were stored at 0 degree C, (crushed ice), 4 degrees C (refrigerator) and 22 degrees C (room temperature). The pressure of oxygen (PO2) fell significantly by 20 minutes at 4 degrees C and 22 degrees C but did not change significantly at 0 degree C for up to 30 minutes. Blood pH, pressure of carbon dioxide (PCO2), and base excess did not change significantly for up to 30 minutes at 4 degrees C and 22 degrees C and up to 60 minutes at 0 degrees C. The effect of air bubbles in the syringe was studied by leaving a single bubble or froth in contact with the blood for one to five minutes in 40 patients. Po2 rose significantly after two minutes'' contact with froth and two minutes'' contact with the air bubble, and PCO2 fell significantly after three minutes'' contact with the air bubble. Size of the bubble had little effect on rates of change. Blood pH, bicarbonate, TCO2, and base excess did not change significantly after up to five minutes'' contact. For accurate estimation of PO2 and PCO2 it is necessary to avoid frothing, to expel all air bubbles within two minutes, and to inject the sample into the machine within 10 minutes or store the syringe in crushed ice. The requirements for blood pH and base excess measurement are less exacting.     

SeP,ApoER2 and megalin as necessary factors to maintain Se homeostasis in mammals

Selenoprotein P (SeP) is an extracellular protein containing ten selenium atoms in the form of selenocysteine, secreted mainly from the liver. About 60% of the whole plasma selenium level is present in SeP, which makes it a useful biomarker of selenium nutritional status. The main functions of SeP are transport and storage of selenium in plasma. It is especially an important protein for the brain, testes and kidneys where the supplementation of the proper amount of Se ensures the synthesis of selenoenzymes with antioxidant properties.Recently, it has been found that SeP uptake in kidneys, testes and brain depends on the apolipoprotein receptor 2 (ApoER2) and lipoprotein megalin receptor (Lrp2). Megalin receptor represents a physiological SeP receptor in kidneys, mediating the re-uptake of secreted SeP from the primary urine. The absence of a functional megalin receptor causes a significant reduction of plasma selenium and the SeP levels as a result of Se excretion. ApoER2 is a SeP receptor in the brain and testes which uptakes Se from the extracellular fluid. Deletion of ApoER2 in mice leads to a lowered selenium level in the brain and testes, neurological dysfunction, production of abnormal spermatozoa, infertility and even death when the subjects are fed a low-selenium diet.     

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated ⁷⁵Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced ⁷⁵Se incorporation from L[⁷⁵Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting ⁷⁵Se incorporation from [⁷⁵Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.     

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).     

The essentiality of sulfhydryl groups to transport in Neurospora crassa.

Unfractionated Escherichia coli B tRNAs have been aminoacylated with selenocysteine by using homologous aminoacyl synthetases. Cochromatography of [³H]cysteyl-tRNA and [⁷⁵Se]selenocysteyl-tRNA on reverse-phase chromatography-5 columns revealed nearly coincident radioactive elution profiles for the two charged tRNAs. Acylation of a mixture of tRNAs with cysteine protected selenocysteine-acceptor activity from inactivation by periodate oxidation. Likewise, preacylation with selenocysteine protected cysteine acceptor from oxidation. Levels of charging with cysteine are reduced about 50% by the presence of a 40-fold excess of selenocysteine. These results indicate that selenocysteine is bound to cysteine-accepting tRNAs, although it does have considerably lower affinity for the ligase than cysteine. The ester linkage of selenocysteyl-tRNA was shown to be somewhat more stable than that of cysteyl-tRNA under the same conditions. These experiments show that selenocysteine can participate in the early steps leading to peptide-bond formation and provide a possible pathway for selenocysteine incorporation into protein.     

Identification of selenocysteine in glutathione peroxidase by mass spectroscopy

A convenient procedure was developed for identifying selenocysteine in selenoproteins by mass spectroscopy, based on formation of the 2,4-dinitrophenyl (DNP) derivative. Pure ovine erythrocyte glutathione peroxidase was reduced with sodium borohydride and reacted with 1-fluoro-2,4-dinitrobenzene at neutral pH under anaerobic conditions in 4 M guanidine. The inactivated enzyme was hydrolyzed with 6 N HCl for 20 h at 110 degrees C under anaerobic conditions. Following extraction of the hydrolysate with benzene, Se-(2,4-dinitrophenyl)selenocysteine in the aqueous phase was separated from non-DNP-amino acids by gel-filtration chromatography and then separated from other water-soluble DNP-amino acids by reversed-phase high-performance liquid chromatography. The Se-(2,4-dinitrophenyl)selenocysteine was converted to Se-methyl-N-(2,4-dinitrophenyl)selenocysteine by the addition of sodium barbital to induce an intramolecular Se leads to N shift (Smiles rearrangement) under anaerobic conditions, in the presence of methyl iodide to trap the liberated selenol group. Following esterification of the product's carboxyl group with methanol and hydrochloric acid, it was subjected to direct probe mass spectroscopy and identified as the methyl ester of Se-methyl-N-(2,4-dinitrophenyl)selenocysteine. This procedure allows selenocysteine to be isolated quite easily as a readily identifiable derivative and has permitted the first identification of a seleno amino acid in a protein by mass spectroscopy.     

Effect on fertility of storing fowl semen for 24 h at 5 degrees C in fluids of different pH.

Improved storage of fowl semen above 0 degree C was achieved by adjusting the pH of the diluent. The fertility obtained with semen stored for 24 h at 5 degrees C in diluents buffered at different pH values was compared with that of semen stored in a basic, unbuffered solution. The most satisfactory result was achieved with diluent buffered at pH 6.8 OR 7.1. Worst fertility was obtained at pH 5.8 and pH 7.4 did not prove very satisfactory. There were indications that the effect of pH under the conditions of the experiment was to regulate metabolism and thereby influence the maintenance of the fertilizing ability of the spermatozoa.     

Exploiting the 21st amino acid-purifying and labeling proteins by selenolate targeting

Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pK(a) and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either gamma-emitting (75)Se or short-lived positron emitters such as (11)C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus.

A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.     

Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30-1 (ATCC 53841)

A thermophilic bacterium was isolated from a hot spring area of Yellowstone National Park. The organism grew optimally at 60–65°C and in the pH range of 6–9. It was characterized as Bacillus sp. In the presence of corn or olive oil (1.0%) as the growth substrate, this Bacillus produced an extracellular lipolytic activity (EC 3.1.1.3). The enzyme activity could be efficiently recovered by ultrafiltration of cell-free culture supernatant. The partially purified lipase preparation had an optimum temperature of 60°C, at an optimum pH of 9.5. It retained 100% of the original activity after being heated at 75°C for half an hour. The half life of the enzyme was 8 h at 75°C. The enzyme retained at least 90% of the original activity after it was incubated at 60°C for 15 h at pH's in the range of 5 to 10.5. The enzyme was active on triglycerides containing fatty acids having a carbon chain length of C16 : 0 to C22 : 0 as well as on natural fats and oils. The enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients. The purified enzyme had an isoelectric point of 5.15 and an approximate molecular weight of 65,000.     

Selenium and selenoproteins in the rat kidney

Kidney tissue contains a high concentration of selenium that is not accounted for by the known selenoprotein glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9). In order to investigate the nonglutathione peroxidase selenium, rats were isotopically labeled with [75Se]selenite over a 10-day period. After this time half of the 75Se in kidney homogenate was found in the particulate subcellular fractions. The kidney lysosomes contained unusually high levels of 75Se, yet they did not contain correspondingly high levels of glutathione peroxidase activity. Two selenoproteins having molecular weights less than 40 000 were resolved by gel filtration from a kidney supernatant fraction. A third selenoprotein exhibited a molecular weight of 75 000. This protein contained one 75 000 molecular-weight subunit, and its selenium was in the amino acid selenocysteine. The 75 000 molecular-weight protein was chromatographically distinct from glutathione peroxidase. In order to determine if these selenoproteins protect against cadmium toxicity, 109CdCl2 was administered to rats that were isotopically prelabeled with 75Se. At 3, 25 and 72 h after 109Cd administration, no 109Cd was associated with selenium-containing proteins. Two of the nonglutathione peroxidase selenoproteins were apparently unique to the kidney.     

The solution and solid state stability and excipient compatibility of parthenolide in feverfew

The objectives of this research were to evaluate the stability of parthenolide in feverfew solution state and powdered feverfew (solid state), and explore the compatibility between commonly used excipients and parthenolide in feverfew. Feverfew extract solution was diluted with different pH buffers to study the solution stability of parthenolide in feverfew. Powdered feverfew extract was stored under 40 degrees C/0% approximately 75% relative humidities (RH) or 31% RH/5~50 degrees C to study the influence of temperature and relative humidity on the stability of parthenolide in feverfew solid state. Binary mixtures of feverfew powered extract and different excipients were stored at 50 degrees C/ 75% RH for excipient compatibility evaluation. The degradation of parthenolide in feverfew solution appears to fit a typical first-order reaction. Parthenolide is comparatively stable when the environmental pH is in the range of 5 to 7, becoming unstable when pH is less than 3 or more than 7. Parthenolide degradation in feverfew in the solid state does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. After 6 months of storage, parthenolide in feverfew remains constant at 5 degrees C/31% RH. However, approximately 40% parthenolide in feverfew can be degraded if stored at 50 degrees C/31% RH. When the moisture changed from 0% to 75% RH, the degradation of parthenolide in feverfew increased from 18% to 32% after 6-month storage under 40 degrees C. Parthenolide in feverfew exhibits good compatibility with commonly used excipients under stressed conditions in a 3-week screening study.     

Aerobic and two-stage anaerobic-aerobic sludge digestion with pure oxygen and air aeration

The degradability of excess activated sludge from a wastewater treatment plant was studied. The objective was establishing the degree of degradation using either air or pure oxygen at different temperatures. Sludge treated with pure oxygen was degraded at temperatures from 22 degrees C to 50 degrees C while samples treated with air were degraded between 32 degrees C and 65 degrees C. Using air, sludge is efficiently degraded at 37 degrees C and at 50-55 degrees C. With oxygen, sludge was most effectively degraded at 38 degrees C or at 25-30 degrees C. Two-stage anaerobic-aerobic processes were studied. The first anaerobic stage was always operated for 5 days HRT, and the second stage involved aeration with pure oxygen and an HRT between 5 and 10 days. Under these conditions, there is 53.5% VSS removal and 55.4% COD degradation at 15 days HRT - 5 days anaerobic, 10 days aerobic. Sludge digested with pure oxygen at 25 degrees C in a batch reactor converted 48% of sludge total Kjeldahl nitrogen to nitrate. Addition of an aerobic stage with pure oxygen aeration to the anaerobic digestion enhances ammonium nitrogen removal. In a two-stage anaerobic-aerobic sludge digestion process within 8 days HRT of the aerobic stage, the removal of ammonium nitrogen was 85%.     

A mouse model for in vivo tracking of the major dust mite allergen Der p 2 after inhalation

Inhaled environmental antigens, i.e. allergens, cause allergic symptoms in millions of patients worldwide. As little is known about the fate of an allergen upon inhalation, we addressed this issue for a major dust mite allergen, Der p 2. First, a model for Der p 2-sensitization was established in C57BL/6 J mice, in which sensitized mice mounted a Der p 2-specific IgE-response with eosinophilic lung inflammation after allergen challenge in the airways. In this model, we applied recombinant Der p 2 carrying a novel C-terminal tetrapeptide Sel-tag enabling labelling with the gamma-emitting radionuclide 75Se at a single selenocysteine residue ([75Se]Der p 2). In vivo tracking of intratracheally administered [75Se]Der p 2 using whole-body autoradiography revealed that [75Se]Der p 2-derived radioactivity persisted in the lungs of sensitized mice as long as 48 h. Radioactivity was also detected in kidneys, liver and in enlarged lung-associated lymph nodes. Interestingly, a larger proportion of radioactivity was found in the lungs of sensitized compared with nonsensitized mice 24 h after intratracheal instillation of [75Se]Der p 2. A radioactive protein corresponding to intact Der p 2 could only be detected in the lungs, whereas [75Se]Der p 2-derived radioactivity was recovered in known selenoproteins both in lung and other organs. Hence, using the recently developed Sel-tag method in a mouse model for Der p 2-sensitization, we could track the fate of an inhaled allergen in vivo. Based upon our findings, we conclude that the inflammatory state of the lung influences the rate of metabolism and clearance of Der p 2. Thus, an allergic response to the inhaled allergen may lead to prolonged retention of Der p 2 in the lung.     

Temperature- and pH-dependence of the oxygen-binding reaction of human fetal haemoglobin.

O2 binding to human haemoglobin F0 was studied at high haem concentrations (3 mM) in the temperature range 15-35 degrees C and in the pH range 6.8-8.7 at 25 degrees C. Comparison with O2 binding to human adult haemoglobin A0 under identical solution conditions reveals striking similarities in the Bohr effect and the enthalpy of oxygenation between the two haemoglobins.