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1.
Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [ 3H-methyl] methionine and [ 35S]methionine into rats, the ratios of [ 3H-methyl]methionine to [ 35S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [ 3H-methyl]- and [ 3S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [ 3H-methyl]methionine would decrease more than that of [ 35S]methionine; i.e., the ratio of [ 3H-methyl]- to [ 35S]methionine in the tissue would decline. The results showed that the ratios of [ 3H-methyl]- to [ 35S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo. 相似文献
2.
The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [ 35S]-methionine or formyl[ 35S]methionyl-tRNA fMet as substrates, and the [ 35S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed. 相似文献
3.
Benzodiazepine GABA-ionophore receptor complex ligands showed persistent modulation of the chloride ionophore, labeled by [ 35S]TBPS, even after receptor complex extensive purification. GABA caused inhibition of [ 35S]TBPS binding, while benzodiazepine agonists increased and benzodiazepine inverse agonists decreased the specific [ 35S]TBPS binding to the purified receptor. When GABA binding sites were occupied by the neurotransmitter benzodiazepine receptor agonists and antagonists reversed their effects clonazepam in fact inhibited and β-carboline ethyl ester increased [ 35S]TBPS binding in the presence of GABA. The antagonist flumazenil showed no effect both in the presence or absence of GABA. 相似文献
4.
The human placenta lacks the enzyme 17-hydroxylase/17-20-lyase, and is thus unable to convert cholesterol into estrogens. Therefore estrogen synthesis of trophoblast cells depends on the supply of precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16-hydroxy-dehydroepiandrosterone-3-sulfate by maternal and fetal blood. To investigate the cellular internalisation of these anionic hydrophilic precursors, the uptake of [ 3H]-/[ 35S]-DHEA-S and [ 3H]-taurocholate by isolated cytotrophoblasts, cells of choriocarcinoma cell lines (JEG-3, BeWo, Jar), BHK and BHK cells transfected with human sterylsulfatase-cDNA (BHK-STS cells) was studied. Furthermore, the activity of sterylsulfatase of these cells in suspension and in corresponding cell homogenate was measured. During the first 5 min of incubation with [3H]-DHEA-S or [35S]-DHEA-S, radioactivity of cytotrophoblasts increased significantly, while radioactivity of JEG-3, Jar, BHK and BHK-STS cells did not increase. Radioactivity of BeWo cells increased slightly. For all cell types, there was no significant difference for uptake of either substrate. During incubation with [3H]-taurocholate, radioactivity of cytotrophoblasts did not increase. Sterylsulfatase activity of cytotrophoblast homogenate was significantly lower than that of cytotrophoblast suspension. Sterylsulfatase activity of BHK-STS, JEG-3 or BeWo cell homogenate was significantly higher than that of the corresponding cell suspension. In BHK and Jar cells sterylsulfatase activity was not detectable. Cytotrophoblasts take up DHEA-S without prior hydrolysis. BHK, BHK-STS, JEG-3, and Jar cells do not take up and BeWo cells slowly take up DHEA-S. In cytotrophoblasts extracellular DHEA-S rapidly gains access to intracellular sterylsulfatase, while in choriocarcinoma and BHK-STS cells access of DHEA-S to sterylsulfatase is limited. Our results indicate, that uptake by cytotrophoblasts is mediated by a carrier which is not expressed in choriocarcinoma or BHK cells and which is different from the known taurocholate-transporting organic anion transporting polypetides. 相似文献
5.
Radiolabel from the methyl groups of serine and methyltetrahydrofolate was readily incorporated into methionine in adult Fasciola hepatica, and a substantial proportion of the label from [ 35S]methionine appeared in cysteine. The data suggest that methionine synthesis is via methyltetrahydrofolate-homocysteine methyltransferase and that there is cysteine synthesis from methionine. Cystathionine-β-synthase and γ-cystathionase activities were demonstrated in homogenates. 相似文献
6.
1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd = 0.45 μM) than carboxyatractyloside or bongkrekic acid. 3. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyactratyloside binding. 5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.
Abbreviations: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone 相似文献
7.
The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT 1A) receptors in rat hippocampus were determined by means of [ 3H]-8-hydroxy-dipropylamino-tetralin ([ 3H]-8-OH-DPAT) and 5′guanosine-(γ-[ 35S]-thio)triphosphate ([ 35S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [ 35S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [ 3H]-8-OH-DPAT binding ( Ki 500 nM) or to reduce the number of specific sites ( Bmax) without affecting Kd. The drug also failed to change the [ 35S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT 1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [ 35S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC 50, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT 1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [ 35S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity. 相似文献
8.
The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [ 35S]sulfate in vivo and [ 35S]3'-phosphoadenosine 5'-phosphosulfate ([ 35S]PAPS) in a cell-free system. In vivo incorporation of [ 35pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [ 35S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage. In a cell-free system, the particulate fraction showed marked incorporation of [ 35pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg 2+and p-chloromercuribenzoic acid. The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage. 相似文献
10.
The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [ 35S]guanosine-5′- O-(3-thio)triphosphate ([ 35S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [ 35S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited. 相似文献
11.
Abstract: The accumulation and utilization of [ 35S]3'-phos-phoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [ 35S]sulfate. [ 35S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [ 35S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [ 35S]PAPS formation was also indirectly followed by incubating slices in the presence of β-naphthol and measuring the levels of [ 35S]β-naphthyl sulfate ([ 35S]β-NS). Whereas [ 35S]PAPS levels rapidly reached a plateau, [ 35S]β-NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [ 35S]PAPS formation in ices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC 50= .10 μM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [ 35S]PAPS accumulation and [ 35S]β-NS'formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N -methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized. 相似文献
12.
By use of membrane preparations and incubation conditions optimized for each binding site, we have characterized the benzodiazepine and ionophore-linked-convulsant/barbiturate modulatory sites within the chick tectal GABA A receptor complex. Using [ 3H]flunitrazepam (FNZ) and [ 35S] t-butylbicyclophosphorothionate (TBPS), respectively, as specific radioligand probes for the two sites, we have found in each case one single population of high-affinity, saturable, specific binding sites. The apparent dissociation constants (K d) show no change during tectal development (9 nM for [ 3H]FNZ, and 25–28 nM for [ 35S]TBPS) while the respective densities of binding sites at saturation ( Bmax) experience in both cases a twofold increase between embryonic day 16 and postnatal day 10. Ligand-specific pharmacological profiles and allosteric interactions between the transmitter and modulatory sites appear to be well preserved in the chick tectal membrane preparations employed in this study. 相似文献
13.
We have measured the rate of equilibration of [ 35S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301–305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNA f that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation. 相似文献
14.
The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [ 35S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, 35S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of 35S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP. 相似文献
15.
ABSTRACT. [ 35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [ 35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [ 3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake. 相似文献
16.
Abstract: We have characterized a high-affinity [ 35S]-glutathione ([ 35S]GSH) binding site in mouse and human spinal cord. [ 35S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [ 35S]GSH binding was saturable, showing a B max of 72 fmol/mg of protein and a K D of 3.0 n M for mouse spinal cord and a B max of 52 fmol/mg of protein and a K D of 1.6 n M for human spinal cord. [ 35S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [ 35S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [ 35S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [ 35S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [ 35S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS. 相似文献
17.
Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [ 35S]GTPγS binding in rat brain. [ 35S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [ 35S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [ 35S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [ 35S]GTPγS binding. Differences in maximal stimulation of [ 35S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [ 35S]GTPγS binding revealed a lower apparent B max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [ 35S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain. 相似文献
18.
We have developed an efficient and selective radioligand, the [ 35S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B 1 receptor. 相似文献
19.
Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [ 35S]methionine and [ 35S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex. 相似文献
20.
Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [ 35S]isethionate (10–20 pmol/g brain wet weight) and [ 35S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [ 35S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [ 14C]GABA was readily metabolized to [ 14C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [ 35S]isethionate (about 1.3 μmol/min/g protein) from [ 35S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool. 相似文献
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