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1.
PROTEIN METHYLATION IN RAT BRAIN IN VITRO   总被引:1,自引:0,他引:1  
Abstract— Protein-methylation activity in various organs of the rat was studied with S-adenosyl-L-[methyl-14C]methionine ([methyl-14C](SAM) as methyl donor. Activity of the enzyme was highest in brain and lowest in liver. Histones comprised approximately 20 per cent of the total radioactivity incorporated, and lysine-rich histone was the most active. Analysis of amino acids of the methylated proteins of rat brain showed arginine to be the amino acid most extensively methylated, but some methylation occurred in lysine residues. An additional [methyl-14C]-labelled amino acid was found near histidine on the amino acid column chromatogram.  相似文献   

2.
Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-14C]putrescine, the specific radioactivity (sra) of the newly formed [14C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-14C]methionine, the other polyamine precursor, significantly higher sra values for [14C]spermidine and [14C]spermine were recorded in the brains of the MSO-treated animals. [14C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-14C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-14C]-methionine. The results of these experiments show both significantly lower sra values for [14C]spermidine and [14C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.  相似文献   

3.
Abstract Monic acid A was isolated from a Pseudomonas fluorescens fermentation in which pseudomonic acid A (PA) was the principal secondary metabolite. [3-14C]3-Hydroxy-3-methyl-glutaric acid (HMG) given early in the idiophase radiolabelled PA (1.1% incorporation), confirming experimentally the putative direct involvement of HMG in the biosynthesis of PA, but contributed relatively insignificant radiolabel to the monic acid extracted from the broth at the end of the fermentation. Ethionine inhibited (80%) PA biosynthesis and correspondingly reduced incorporation of [14C]HMG. In contrast, ethionine increased incorporation of [methyl-14C]methionine into PA and enhanced specific radioactivity of the antibiotic 8-fold. Ethionine inhibition of secondary metabolite methylations did not divert pseudomonate biosynthesis to give unusual analogues, implying that methylation of a putative pentaketide precursor of the monate moiety forms a vital intermediate of the pseudomonate pathway, but caused a new [14C]HMG-derived polar metabolite of biosynthetic interest to become evident.  相似文献   

4.
The role of methionine as a precursor in mugineic acid (MA) biosynthesis was studied by feeding 15N-ammonium sulfate, 14C-amino acids, and [1-14C, 15N]-methionine to iron-deficient barley roots ( Hordeum vulgare L. cv. Minorimugi), grown hydroponically. The incorporation of isotopes into amino acids was also examined. Methionine appears to be the most efficient precursor of the mugineic acid family (MAs) of phytosiderophores; homoserine was also incorporated into the MAs, but other amino acids such as glutamate, alanine, and γ-amino butyric acid did not act as precursors of MAs. Carbon-14 and 15N of methionine were incorporated into MAs. This specific incorporation of 14C and 15N indicated that the nitrogen atoms of MAs were derived from two molecules of methionine. It is suggested that deoxymugineic acid (DMA) is probably the first phytosiderophore to be synthesized on the biosynthetic pathway of MAs.  相似文献   

5.
Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[14C]arginine and the NOS inhibitor [14C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[14C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N G-methyl- l -arginine, N G-amino- l -arginine, and N 5-imidoethyl- l -ornithine. l -[14C]Arginine uptake was not inhibited by aminoguanidine or N G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [14C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[14C]arginine.  相似文献   

6.
Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [14C]glycine, [3H]GABA, l -[14C]glutamate. l -[14C]aspartate, l -[14C]serine, l [14C]threonine or l -[3H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [14C]glycine, [3H]GABA, l -[14C]glutamate and l -[14C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[14C]serine, l -[14C]threonine, l -[14H]leucine, [14C]mannitol or [14C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.  相似文献   

7.
Abstract— The uptake of [14C]GABA, [14C]taurine, [3H] β -alanine and [14C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [14C]Taurine and [3H] β -alanine uptake increased whereas [14C]GABA uptake decreased with increasing slice size. [14C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [3H] β -alanine and [14C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.  相似文献   

8.
Abstract— By using a combination of subcutaneous and intraventricular injections of [14C]uridine and [3H]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 [14C]uridine and [3H]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 [14C]uridine and [3H]-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   

9.
Abstract— The formation of histamine in brain was studied in mice injected with l -[14C]-histidine (ring 2-14C) intravenously (i.v.) or intracerebrally; [14C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[14C]histamine and [14C]-methyl-histamine in mice given (1) [14C]histamine i.v., (2) [14C]histamine intracerebrally, and (3) l -[14C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.  相似文献   

10.
Abstract— Unilateral sympathetic decentralization of the superior cervical ganglion of rats was performed 3 days prior to the experiments. A two-compartment kinetic model was proposed to describe the effect of decentralization on (1) the uptake of a nonphysiological amino acid from plasma to the submaxillary gland and (2) the incorporation of a physiological amino acid from precursor pool into protein. The calculations based on the model showed that the fractional rate constant for efflux of the nonphysiological amino acid, α-[3-14C] aminoisobutyric acid, was greater in the decentralized than in the normal gland. However, efflux rate was equal in the two glands because the extrapolated zero time value of the initial concentration was greater in the normal gland.
The labelled physiological amino acid, [14C]leucine, was used in initial experiments to assess turnover rate of the gland proteins but it was rapidly metabolized to many other radioactive compounds. Therefore, arginine[14C]guanido was employed-arginine being the only labelled amino acid found after injection. Since the steady state content of submaxillary gland proteins was not changed but the fractional rate constant of conversion of free arginine into protein (kp) was greater in the decentralized gland (kp= 0-40 h_l) than in the normal (kp= 0-27 h−1), we can conclude that decentralization increases protein turnover rate; thus, assuming that arginine[14C]guanido is homogeneously distributed in the tissue pools of free arginine, the rate of protein turnover is greater in the sympathetically decentralized gland than in the normal.  相似文献   

11.
Isoprenoid biosynthesis in bacteria: Two different pathways?   总被引:4,自引:0,他引:4  
Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [14C]acetyl-CoA or [14C]hydroxymethylglutaryl-CoA to [14C]mevalonic acid. Furthermore, [14C]mevalonic acid, [14C]mevalonate-5-phosphate and [14C]mevalonate-5-pyrophosphate were metabolized to [14C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.  相似文献   

12.
β -Alanine betaine is an osmoprotective compound accumulated by most members of the plant family Plumbaginaceae. Leaf and root tissues of Limonium latifolium known to accumulate β -alanine betaine readily convert supplied β -alanine to β -alanine betaine. To identify the intermediates and the enzymes involved in β -alanine betaine synthesis, radiotracer experiments using [ 14 C] formate were employed. These studies demonstrate that β -alanine betaine is synthesized from β -alanine via N -methyl and N,N- dimethyl β -alanines. A rapid and sensitive radiometric assay was developed to measure N -methyltransferase (NMT) activities by using [methyl-14C] or [methyl-3H] S -adenosyl- l -methionine (AdoMet) as the methyl donor. Leaf extracts from β -alanine betaine accumulators – Armeria maritima , L. latifolium and L. ramosissimum – had detectable NMT activities while none were found in L. perezii , a species that does not accumulate β -alanine betaine. The NMT activities were further characterized from the leaves of L. latifolium . The activities had a pH optimum of 8.0, were soluble and inhibited by S -adenosyl- l -homocysteine. Extractable activities were similar from plants grown under control and salinity stress conditions. Radiolabeling with [ 14 C] l -aspartic acid indicated that, unlike in bacteria, decarboxylation of l -aspartic acid is not the source of β -alanine in the Plumbaginaceae.  相似文献   

13.
Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent Km = 0.64 m m ), KC1 (apparent Km = 14 m m ) and MgCl2 (optimal concentration range 4-15 m m ). The apparent Km for phenylalanine was 2.9 m m . [14C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [14C]phenylalanine and [14C]tyrosine when incubated simultaneously or successively. The Km and Kt (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [14C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [14C]Phenylalanine and [14C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.  相似文献   

14.
Abstract— Incorporation of [14C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[14C]Tyrosine was released from assembled and non-assembled [14C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[14C]tyrosine preparation in the presence of CaCl2 at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [14C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[3H]Phenylalanine was released from a preparation of tubulinyl-[3H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[14C]tyrosine and tubulinyl-[3H]phenylalanine was partially assembled, the ratio of 14C/3H found in the microtubules was the same as in the non-assembled tubulin fraction.  相似文献   

15.
The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N 6[8-I4C]furfuryladenine or of [8-14C]adenine to the root system of decapitated plants and to cultured excised buds. When N 6[8-14C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-14C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-14C]adenine or N 6[8-14C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N 6[8-I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-14C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N 6 position of the adenine base.  相似文献   

16.
Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [14C]oleoyl-CoA or [14C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [14C]oleoyl-CoA or [14C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[14C]18:1, 16:0/[14C]18:1 and 18:1/[14C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [14C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [14C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[14C] Γ -18:3, 18:1/[14C] Γ -18:3 and 16:0/[14C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.  相似文献   

17.
IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY l-DOPA   总被引:3,自引:2,他引:1  
Abstract— A study has been made of the effect of a single intraperitoneal dose of l -DOPA on the in vivo metabolism of [14C]leucine and [14C]lysine by the brain, and on their uptake into brain protein. Administration of 500 mg DOPA/kg to 40-g rats raised the concentrations of several free amino acids; the only amino acid which underwent a statistically significant increment was alanine. Intracisternally-injected [U-14C]leucine was rapidly metabolized to other labelled compounds; DOPA administration did not influence significantly the rate of its metabolism. No similar metabolic change was observed after administering [U-14C]lysine intracisternally.
Incorporation of [14C]leucine and [14C]lysine into total brain protein was significantly reduced 45 min after DOPA administration. There was also depression of the uptake of labelled amino acid into a supernatant fraction, obtained by high speed centrifugation of the brain homogenate, and into brain microtubular protein (tubulin). Reduced amino-acid incorporation into brain proteins observed 45 min after l -DOPA injection coincided with extensive disaggregation of brain polyribosomes. At 120 min after DOPA treatment, disaggregation was no longer significant and there was a smaller depression in labelled amino aicd incorporation, which disappeared completely 240 min after l -DOPA injection. It is concluded that disaggregation of brain polysomes following DOPA treatment is an accurate reflection of a change in the intensity of brain protein synthesis in vivo.  相似文献   

18.
Lumiflavin and Lumichrome Transport in the Central Nervous System   总被引:1,自引:0,他引:1  
Abstract: The transport of the lipid-soluble sugarless flavins, [14C]lumiflavin and [14C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [14C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [14C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [14C]lumiflavin and [14C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [14C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [14C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [14C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [14C]lumiflavin, almost all of the [14C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [14C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.  相似文献   

19.
Abstract— [14C]Nipecotic acid was accumulated in isolated desheathed rat dorsal root ganglia by a saturable process with K m= 48.8 μ m and V max= 2.2 nmol/g/min. The concentration of l -2.4-diamino-butyric acid required to inhibit the uptake of nipecotic acid by 50% was three times the concentration of β-alanine required to do the same. Light microscopic autoradiography indicated that the sites of uptake of [14C]nipecotic acid were principally confined to satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.  相似文献   

20.
Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol- O -methyltransferase activity is described. The method is based on the conversion of catechol to [3H]guaiacol by catechol- O -methyltransferase in the presence of Mg2+, adenosine deaminase and S -adenosyl l -[methyl-3H]methionine. Incubation and direct extraction of [3H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol- O -methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.  相似文献   

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