共查询到20条相似文献,搜索用时 15 毫秒
1.
Beate Averhoff Garabed Antranikian Gerhard Gottschalk 《FEMS microbiology letters》1986,33(2-3):299-304
Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [32 P]orthophosphate, [14 C]inosine or [14 C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the 32 P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the 14 C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate. 相似文献
2.
This report demonstrates that during the torpor phase of hibernation, hamsters utilize 14 C and 13 C glucose in torpor-specific brain metabolic pathways. Microdialysis of 14 C glucose into the striatum rapidly induced a steady state labeling of extracellular fluid (ECF) lactate and labeling of tissue GABA, glutamate, glutamine, and alanine in ipsilateral and contralateral striata. The same tissue metabolites were labeled in cortex, hypothalamus, and brainstem after microdialysis of 14 C lactate into the lateral ventricle. Serine, aspartate, glycine, taurine, tyrosine, and methionine were not synthesized from glucose or lactate during torpor. ECF levels of amino and organic acids were low and unchanging during torpor and increased late during arousal to cenothermia. Labeled intracellular 14 C GABA and glutamate were not communicated to the striatal ECF or ventricular space during torpor. 13 C NMR demonstrated rapid formation of lactate and functional tricarboxylic acid cycles in GABAergic and glutamatergic neurons, and enrichment of glutamine and alanine after i.v. 13 C glucose. Large changes in tissue levels of amino acids occur prior to or during entrance into torpor but not during torpor. It is proposed that cerebral intracellular dehydration, the enlargement of ECF and the biochemistries associated with brain water homeostasis may have a role in regulating hibernation. 相似文献
3.
CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM 总被引:4,自引:1,他引:3
Christo Goridis Muhammed A. Joher Marierose Hirsch Melitta Schachner 《Journal of neurochemistry》1978,31(2):531-539
Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase-catalysed radioiodination and by their interaction with an anti-mouse cerebellum antiserum (anti-NS-4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non-nervous tissues showed a different gel pattern. Immuno-precipitates of solubilizcd surface-iodinated cerebellar cells with anti-NS-4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 103 and 145 × 103 . These proteins were also biosynthetically labeled with [3 H]leucine. The 145 × 103 molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 103 molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 103 . 相似文献
4.
Soluble and microsomal proteins synthesized at various stages of oogenesis in Xenopus laevis were compared by 3 H and 14 C dual-isotope labelling with subsequent mixing and analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results indicated that the proteins labelled in all the stages of oogenesis studied are remarkably similar. However, the patterns in the small and medium-sized oocytes are more similar to one another than to the patterns characteristic of large oocytes. The greatest differences were found when comparing the microsomal proteins.
The labelling patterns of oocyte proteins in vitro were not significantly different from the in vivo patterns for the stages of oogenesis studied. These results indicate that (1) there is little quantitative contribution of proteins to either the soluble or microsomal fractions from extra-oocytic sources in vivo and (2) the in vitro system itself has little effect on the labelling patterns over the incubation period. 相似文献
The labelling patterns of oocyte proteins in vitro were not significantly different from the in vivo patterns for the stages of oogenesis studied. These results indicate that (1) there is little quantitative contribution of proteins to either the soluble or microsomal fractions from extra-oocytic sources in vivo and (2) the in vitro system itself has little effect on the labelling patterns over the incubation period. 相似文献
5.
Denise A. Figlewicz Richard H. Quarles David Johnson Gary R. Barbarash Nancy H. Sternberger 《Journal of neurochemistry》1982,37(3):749-758
Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3 H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3 H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14 C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS. 相似文献
6.
Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes 总被引:11,自引:10,他引:1
Abstract: To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K+ -evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 m M K+ . In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, five sequential K+ -depolarizations caused a decremental release of preformed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [14 C]ACh was unaltered while new ACh was being synthesized from exogenous [3 H]choline. Since the evoked release of [3 H]ACh was maintained while that of [14 C]ACh was decreasing, the [3 H]ACh/[14 C]ACh ratio in perfusate increased with each successive depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release. 相似文献
7.
We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3 H]ethanolamine or [3 H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3 H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3 H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors. 相似文献
8.
Abstract— The synthesis of γ-aminobutyric acid (GABA) in mouse brain was decreased by treatment of the animals with pyridoxal phosphate- γ-glutamylhydrazone, an inhibitor of glutamate decarboxylase in vivo. Under these experimental conditions the following parameters were studied: (1) the incorporation of labeled leucine in vivo , into protein of brain subcellular fractions; (2) the brain polysome profile; (3) the incorporation of labeled leucine into protein in vitro , in ribosomal preparations isolated from brain tissue. In other experiments, GABA synthesis was also decreased in brain cortex slices by preincubation with aminooxyacetic acid. The incorporation of [3 H]leucine or [14 C]leucine into protein in these slices was studied, and samples from the proteins were subjected to acrylamide-sodium dodecylsulfate gel electrophoresis. Radioactivity was counted in slices of the gel. The results of the experiments in vivo and in vitro indicate that the previously reported decrease of protein synthesis induced by an inhibition of GABA synthesis affects proteins of all subcellular fractions and all populations of protein as separated by gel electrophoresis. The polysome profile from brains of mice with decreased GABA synthesis was similar to that of control mice. This result differs from that found when brain protein synthesis is inhibited by dopamine and serotonin. 相似文献
9.
Abstract: Tubulin was tyrosinated in slices and in extracts of brain of rats of 3, 25, and 120 days of age by successive incorporation of [14 C]tyrosine and [3 H]-tyrosine, respectively. The release of the incorporated amino acid was measured by using tubulinyl-tyrosine carboxypeptidase, carboxypeptidase A, and tubulin-tyrosine ligase. With the carboxypeptidases no differences in either the rates or the extents of the release of tyrosine between these two differently labeled tubulins were found. Differences were found when the detyrosination was catalyzed by the ligase and these were attributed to a higher inactivation of tubulin labeled in slices than of that labeled in extracts. 相似文献
10.
Abstract: Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [13 C7 ]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [2 H3 ]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood-brain barrier were calculated from the extraction fraction of [13 C7 ]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [2 H3 ]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 n M ) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2-fold) and caused a rise in homogenate (10.8-fold) and ECF (18.5-fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246-fold) and ECF (66-fold) QUIN levels occurred because of an increase in local synthesis rate (146-fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations. 相似文献
11.
SUBCELLULAR FRACTIONATION STUDIES RELATED TO THE PROCESSING OF NEUROSECRETORY PROTEINS IN APLYSIA NEURONS 总被引:1,自引:0,他引:1
Abstract— Neurosecretory cells (bag cells and R3–14 neurons) in the abdominal ganglion of Aplysia californica were 'pulse-chased' in [3 H]leucine and comparisons of the labeled protein profiles from the total cell homogenate versus a crude 'neurosecretory granule' fraction on acid-urea polyacrylamide gels were made, The data provides indirect support for the hypothesis that some of the post-translational processing of the neurosecretory proteins occurs intragranularly (L oh et al , 1975). In the case of the Bag cells the initial processing of the 29,000 daltons precursor appears to occur extragranularly, possibly in the rough endoplasmic reticulum cisternae. 相似文献
12.
Henry A. Lester Michael L. Steer Daniel M. Michaelson† 《Journal of neurochemistry》1982,38(4):1080-1086
Abstract: Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [32 P]NAD+ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of Mr = 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcel-lular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with Mr = 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nu-cleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes. 相似文献
13.
Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)+ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T1 ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)+ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)+ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)+ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)− RNA was larger than 18s. The proportion of poly(A)+ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults. 相似文献
14.
Abstract— Mice were injected intracerebrally with mixtures of [3 H]fucose and [14 C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [3 H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [14 C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase. 相似文献
15.
Abstract— By using a combination of subcutaneous and intraventricular injections of [14 C]uridine and [3 H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [14 C]uridine and [3 H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [14 C]uridine and [3 H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction. 相似文献
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [
16.
Dean C. Crick Julie Suders Catherine M. Kluthe Douglas A. Andres Charles J. Waechter 《Journal of neurochemistry》1995,65(3):1365-1373
Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 n M ), pig brain (IC50 = 21 n M ), and C6 glioma cells (IC50 = 35 n M ) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis -isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [3 H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21 ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3 H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3–5 µ M ) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F-P-P. 相似文献
17.
CITRATE AS THE PRECURSOR OF THE ACETYL MOIETY OF ACETYLCHOLINE 总被引:13,自引:12,他引:1
Abstract— Rat brain cortex slices were incubated with glucose labeled with either 3 H or 14 C in the 6-position. The 3 H/14 C ratios and the incorporation of radioactivity into lactate, citrate, malate and acetylcholine were determined. While the 3 H/14 C ratio of lactate was close to that of glucose, the ratios in the acetyl moiety of acetylcholine and the acetyl (C-4,5) portion of citrate decreased in a similar proportion. This was interpreted as indirect evidence for the participation of citrate as a precursor to the acetyl moiety of acetylcholine. Two inhibitors of the citrate cleavage pathway: n -butylmalonate, an inhibitor of citrate transport and (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase were studied for their effect on acetylcholine synthesis. N -butylmalonate (10 mM) and (-)-hydroxycitrate (7.5 mM) led to a decrease in the per cent of 14 C recovered as acetylcholine. In each instance the 3 H/14 C ratio in acetylcholine was higher in the presence of inhibitor while the corresponding ratios in lactate and citrate (C-4.5) remained unchanged. From the results, it is suggested that citrate is involved in the transport mechanism of acetyl units from its site of synthesis in mitochondria to the site of acetylcholine synthesis in the cytosol. 相似文献
18.
Abstract— Guinea pig neocortical tissues were incubated with [14 C]adenine, dispersed in cold isotonic sucrose and subcellular fractions prepared by centrifugation. Some 98 per cent of the assimilated 14 C was found as acid-soluble nucleotides in the incubated tissues. In primary fractions obtained by differential centrifugation, about 60 per cent of the [14 C]-nucleotides were in supernatant fractions, in distinction to ATP of which the greatest molar quantity (61 per cent of that in the dispersion) was in the crude mitochondrial fraction. When the crude mitochondrial fraction was separated by density gradient centrifugation, most 14 C was found in synaptosomal fractions and about 85 per cent of this 14 C was adenine nucleotides.
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of14 C subsequently found in nucleotides (collectively) in the supernatant fraction, and increased their inosine and hypoxanthine. Stimulation increased the tissue's cyclic AMP but a preferential localization for this was not established. Results are tentatively interpreted in terms of liberation of an adenine derivative on excitation, and its action or reuptake at a tissue component different from that from which it was liberated. Fractionation of tissues which had been incubated with both [14 C]-adenine and [3 H]adenosine suggested that of the two compounds, more adenosine was taken up by synaptic regions in preference to other cellular regions of the tissue. 相似文献
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of
19.
Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [35 S]isethionate (10–20 pmol/g brain wet weight) and [35 S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [35 S]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. [14 C]GABA was readily metabolized to [14 C]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 [35 S]isethionate (about 1.3 μmol/min/g protein) from [35 S]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
20.
Abstract Cell contents of Clostridium sphenoides , labeled with [32 P]orthophosphate under strict anaerobic conditions, were analyzed by two-dimensional gel electrophoresis. Autoradiography of these gels demonstrated the presence of at least 15 32 P-labeled protein species, of which M r and iso-electric point were determined. Treatment of the radioactively labeled cell contents with alkaline phosphatase and acid phosphatase showed that all these proteins were modified by phosphorylation. These findings demonstrate for the first time the presence of phosphorproteins in a strictly anaerobic bacterium. 相似文献