共查询到20条相似文献,搜索用时 31 毫秒
1.
Rudolf Geyer Hildegard Geyer Gerhard Hunsmann Josef Schneider Stephan Stirm 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(3):491-501
The sugar composition of the surface glycoprotein from Friend murine leukemia virus was determined by gas-liquid chromatography of the alditol acetates and by the thiobarbituric acid method, respectively. N-Acetylglucosamine, mannose, galactose, sialic acid and fucose were found in a molar ratio around 15.2:11.6:7.4:3.3:1.0. Ten ogligosaccharide fractions were obtained from glycoprotein preparations by a suitable sequence of degradation (with pronase, endo-β-N-acetylglucosaminidase H, neuraminidase, and by hydrazinolysis) and separation procedures (concanavalin A-affinity chromatography and gel filtration). The qualitative sugar composition of these fractions was analyzed by in vivo labelling with D-[6-3H]glucosamine, D-[2-3H]mannose, D-[6-3H]galactose, or L-[6-3H]fucose, and their molecular weights were estimated from the gel elution volumina. Four fractions of N-glycosidically linked oligosaccharides of the oligomannosidic (‘high mannose’) type oligomannosidic7-oligomannosidic10, about seven to ten sugar residues), two of the mixed (M11 and M12), and four of the N-acethyllactosaminic (‘complex’) type (N-acetyllactosaminic9, probably nine sugar residues; (N-acetyllactosaminica-N-acetyllactosaminicc, size unknown) were thus identified. 相似文献
2.
Summary. A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was
synthesized and tritiated by addition of 3H2 to double bond of 3,4-dehydroproline residue. 3H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl β-hydrogens.
In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [3H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[3H] Pro-Ala-OH, [3H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[3H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[3H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro5-[3H] Pro-OH and H-Asp-[3H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and
ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed. 相似文献
3.
Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis 总被引:1,自引:1,他引:0
Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [3H]Inositol 1,4,5-trisphosphate [[3H]Ins(1,4,5)P3] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ~140% of basal values. [3H]Inositol 1,4-bisphosphate [[3H]Ins(1,4)P2] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [3H]inositol 1,3,4-trisphosphate [[3H]-Ins(1,3,4)P3], [3H]inositol 1,3-bisphosphate, and [3H]-inositol 4-monophosphate ([3H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca2+, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [3H]Ins(1,4,5)P3 was rapidly metabolized (t1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t1/2 values of 113, 133, 104, and 66 s for [3H]Ins(1,3,4)P3, [3H]Ins(1,4)P2, an unresolved mixture of [3H]inositol 1- and 3-monophosphate ([3H]Ins1/3P), and [3H]Ins4P, respectively. The calculated turnover rate of [3H]Ins(1,4,5)P3 was sufficient to account for the turnover of the combination of both [3H]Ins(1,4)P2 and [3H]Ins(1,3,4)P3 but not that of [3H]Ins1/3P or [3H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca2+-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate. 相似文献
4.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(1):17-22
The thioesterase activity of porcine pancreatic phospholipase A2 has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [3H]palmitic acid from [3H]palmitoyl-acyl carrier protein. 相似文献
5.
G Shanmugam 《Biochemical and biophysical research communications》1976,70(3):818-823
Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 106) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 106 and 1.5 × 106. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [3H]poly(A)·poly(U) but not [3H]poly(C) or [3H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage. 相似文献
6.
AKR MuLV 70S RNA was separated on Poly(U)-Sepharose into poly(A) and non-poly(A) containing 34S subunits. The ratio of the two fractions was 2:1, respectively. Both fractions were hybridized to AKR MuLV [3H]cDNA, and the hybrids were assayed by nuclease S1 and cesium sulfate centrifugation. The poly(A) and non-poly(A) subunits hybridized to [3H]cDNA to the same extent (80%), with identical values; and the hybrids of both fractions had identical Tm values (81°C in 0.15 M NaCl). These results demonstrate that the poly(A) and non-poly(A) containing subunits of the AKR genome have identical or very similar base sequences in the heteropolymeric regions. 相似文献
7.
Methods are described for the synthesis of [3H]chloramphenicol and derivatives labeled on carbon 1 of the propanediol side chain, with a specific activity of about 2 mCi/μmol. The labeling step involves the reduction of the I-oxo derivative of N-acetyl chloramphenicol base by KB3H4 to produce a mixture of the d (?) threo- and d (?) erythro-diastereoisomers, since carbon 1 is an asymmetric carbon atom. The two isomers were separated by thin-layer chromatography after acetylation of the two free hydroxyls. After hydrolysis of the three acetyl groups, the biologically active d (?) threo-[1 ? 3H]chloramphenicol base was converted to chloramphenicol. Modification of the above procedures allows the rapid and simple preparation of the mixed d (?) threo- and d (?) erythro-isomers of [1 ? 3H]chloramphenicol. This mixture can be used where the presence of the inactive d (?) erythro-isomer of chloramphenicol is not important. The modified procedure also allows the preparation of the mixed isomers of [1 ? 3H]chloramphenicol base and of chloramphenicol analogs. Attempts to prepare a 3-aldehyde derivative of chloramphenicol were not successful. If this could be done, reduction of this derivative with KB3H4 would produce the correct isomer of chloramphenicol since carbon atom 3 on the propanediol side chain is not an asymmetric carbon atom. 相似文献
8.
《Insect Biochemistry》1990,20(2):149-156
The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-3H]valine, [4,5-3H]isoleucine and [3,4-14C2]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-14C2]- and [2,3-14C2]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-13C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-13C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-13C3]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation. 相似文献
9.
The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [3H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [3H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [3H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [3H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [3H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P1 digestion reduced 99% of the [3H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [3H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [3H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [3H]BV-araU is incorporated inside viral DNA strands in infected cells. 相似文献
10.
J. Bodennec G. Brichon O. Koul J. Portoukalian G. Zwingelstein 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,125(4)
Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([3H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [3H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments. 相似文献
11.
A. M. Cesura R. Imhof B. Takacs M. D. Galva G. B. Picotti M. Da Prada 《Journal of neurochemistry》1988,50(4):1037-1043
Abstract: [3H]Ro 16–6491 [N-(2-aminoethyl)-p-chloroben-zamide HCl], a reversible “mechanism-based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO-B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO-B inhibitor /-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO-B, e.g., by /-deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO-A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO-B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO-B activity whereas MAO-A activity was unaffected. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO-B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO-B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491-MAO-B complex results in the formation of a stable adduct with the amino acid chain of the MAO-B or with its prosthetic group, FAD, remains to be elucidated. 相似文献
12.
The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of 3HOH from D-[5-3H]glucose and 14C-labelled CO2, acidic metabolites and amino acids from D-[U-14C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types. 相似文献
13.
Rafael Brito Mariana Rodrigues Pereira Roberto Paes‐de‐Carvalho Karin da Costa Calaza 《Journal of neurochemistry》2012,123(2):239-249
Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N6‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [3H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [3H]DPCPX binding, and reduced A2A receptors. The A2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [3H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [3H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A2A receptors modulators. 相似文献
14.
Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N-ArPE (p < 0.05) (1.43-fold for [3H]AnNH and 1.65-fold for [3H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N-ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [3H]AnNH and [3H]N-ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and Gi/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH. 相似文献
15.
The influence of photoperiod on the metabolism of GA20 in Salix pentandra was studied by feeding [3H]-GA20 to seedlings which had been grown previously under long day (LD) or short day (SD) conditions. After 48 h in LD or SD, metabolites were separated on sequential, silica gel partition columns and reversed-phase C18 HPLC. The principal metabolite co-chromatographed with [3H]-GA1 and this conversion was confirmed by feeding [2H]-GA20, which was converted to [2H]-GA1 as identified by gas chromatography-selected ion monitoring. Chromatographic evidence also indicated the minor conversion of [3H]-GA20 to [3H]-GA8 (via [3H]-GA1) and trace conversion to [3H]-GA29 (GAs A1.8,20.29 are native in Salix). Ethyl acetate-insoluble [3H] metabolites were formed and could be cleaved by cellulase to release putative [3H]-GA20 and [3H]-GA1 suggesting the conversion to glucosyl conjugates of these GAs. Metabolism of [3H]-GA20 was slightly more rapid in plants previously grown under LD than SD, an effect which reflected the generally increased shoot growth under LD. However, altering the photoperiod after [3H]-GA20 addition had only a slight effect on the metabolism of [3H]-GA20 in Salix seedlings. This indicates that the conversion of GA20 to GA1 is not a controlling step in the photoperiodic regulation of growth cessation in Salix. 相似文献
16.
Opiate Receptor-Mediated Inhibition of Catecholamine Release in Primary Cultures of Bovine Adrenal Chromaffin Cells 总被引:10,自引:5,他引:5
Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg6,Phe7], and the synthetic peptide [d -Ala2,Me-Phe4,Met(O)s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC30 varying from 10?7 to 1 × 10?6M. The effect was stereospecific because levorphanol (IC30= 7.5 × 10?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala2, d -Leu5]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC50= 5 × 10?7M) and naloxone (IC50= 10?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [3H]etorphine, [3H]dihydromorphine, [3H-d -Ala2,d -Leu5]enkephalin, [3H]ethylketazocine, and for [3H]N-allylnormetazocine, [3H]diprenorphine, and [3H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [3H]etorphine and [3H]diprenorphine was 10–70 times higher than the number of sites measured with the other 3H ligands. The rank order of potency of these compounds for the displacement of [3H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg6,Phe7] modulates the function of the nicotinic receptor in adrenal chromaffin cells. 相似文献
17.
The effects of hyperbaric oxygen on uracil nucleotide metabolism in B104 rat neuroblastoma cells were investigated. Cells exposed to 10 atm O2 for 4 h incorporated markedly less [3H]uridine into the acid-soluble fraction and RNA compared to cells kept in ambient air. The acid-soluble fraction of the oxygen-treated cells contained less total [3H]uridine phosphates ([3H]UMP + [3H]UDP + [3H]UTP) than air-treated cells. Uridine kinase activity, assayed in cytosolic extracts from cells exposed to 10 atm O2 for 4 h, was decreased by 46% compared to the air controls. The reduced enzyme activity which appears to account for the depressed [3H]uridine incorporation, may contribute to the lethal effects of oxygen in these cells.Abbreviations DMEM
Dulbecco's Modified Eagle's Medium
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid 相似文献
18.
M. V. Catania Z. Hollingsworth J. B. Penney A. B. Young 《Journal of neurochemistry》1993,60(1):236-245
Abstract: Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A2 (PLA2) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA2 pretreatment induced a significant increase in α-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [3H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [3H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [3H]glutamate binding site were also increased by PLA2 pretreatment. [3H]Kainate binding and NMDA-sensitive [3H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA2 effect was reversed by EGTA, the PLA2 inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA2. Arachidonic acid failed to mimic the PLA2 effect on metabotropic binding. These results indicate that PLA2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS. 相似文献
19.
Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [3H]tryptophan, [3H]methionine and [3H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (Mr 12 000) and H (Mr 3 000) highly labelled with [3H]tryptophan, lower with [3H]methionine but not with [3H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg2– soluble fractions which suggests that, similar to the fractions I (Mr below 3 000) and B (Mr 120 000) described previously (5), these fractions may be associated with active transcribed genes. 相似文献
20.
Zuzana Dostalova Xiaojuan Zhou Aiping Liu Xi Zhang Yinghui Zhang Rooma Desai Stuart A. Forman Keith W. Miller 《Protein science : a publication of the Protein Society》2014,23(2):157-166
Gamma‐aminobutyric acid type A receptors (GABAARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABAARs determine their function and pharmacological profile. GABAARs are heteropentamers of subunits, and (α1)2(β3)2(γ2L)1 is a common subtype. Biochemical and biophysical studies of GABAARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABAAR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)3GK–1D4 GABAAR. These cells achieved expression levels of 70–90 pmol [3H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [3H]flunitrazepam to [3H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABAARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ~30%. Typical purifications yielded 1.0–1.5 nmoles of [3H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [3H]muscimol binding were maintained in the purified state. 相似文献