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1.
Yoshiaki Takahashi Kikuo Ogata 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,697(1):101-112
The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [14C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·104 and 2.1·104 were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with or , and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits. 相似文献
2.
Carlos E. Argaraña Carlos A. Arce Hector S. Barra Ranwel Caputto 《Archives of biochemistry and biophysics》1977,180(2):264-268
In vitro incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [14C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [14C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [14C]tyrosine. The α and β subunits of tubulin from animals that received [14C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [14C]amino acids was injected, the radioactivity ratio of subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo. 相似文献
3.
γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. , 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. , 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-14C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the 14C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme. 相似文献
4.
The mode of incorporation of 6-benzylaminopurine into tobacco callus transfer ribonucleic Acid: a double labeling determination
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Cytokinin-dependent tobacco (Nicotiana tabacum) tissue was grown in the presence of N6-benzyladenine (6-benzylaminopurine) labeled in the phenyl ring with 3H and in the 8-position of the purine ring with 14C. The 3H/14C ratio of N6-benzyladenosine recovered from labeled tobacco callus transfer RNA preparations was compared with the corresponding ratio for N6-benzyladenine supplied in the medium. By this means, with suitable controls, the incorporation of the synthetic cytokinin, N6-benzyladenine, into the transfer RNA of tobacco callus tissue was shown to involve the intact moiety. The observed level of incorporation was up to one N6-benzyladenosine molecule per 104 transfer RNA molecules. 相似文献
5.
A proteinase (called Proteinase I) present in myxamoebae of the cellular slime mold, , was labeled with [32P] by growth of cells on media containing [32P] orthophosphate. The labeled proteinase was purified to apparent homogeneity and characterized by dissociation chromatography and quantitative immune-precipitin analysis. Based upon the results of these studies it was concluded that phosphoryl moieties were tightly associated (presumably covalently bonded) with the polypeptide subunits of Proteinase I. 相似文献
6.
In vivo biosynthesis of -linolenic acid in plants 总被引:6,自引:0,他引:6
C G Kannangara B S Jacobson P K Stumpf 《Biochemical and biophysical research communications》1973,52(2):648-655
[1-14C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of and . In these systems the [14C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C1–9 and the C10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [14C] was localized in the C1–9 fragment. While [1-14C]oleic acid was converted by whole cells of to [1-14C]linoleic and [1-14C]linolenic acids, [U-14C]oleic acid yielded [U-14C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid. 相似文献
7.
W.J. Burrows 《Biochemical and biophysical research communications》1978,84(3):743-748
Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing 3H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl--zeatin, -zeatin, N6-(Δ2-isopentenyl) adenosine and N6-(Δ2-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N6-(Δ2-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of -zeatin and ribosyl--zeatin. 相似文献
8.
J S Hothersall N Baquer A L Greenbaum P McLean 《Archives of biochemistry and biophysics》1979,198(2):478-492
The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-3H]glucose and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (14CO2 yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (14CO2 yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed. 相似文献
9.
The rate of 14C-leucine incorporation into protein has been examined in rat kidney tissue. The presence of a marked gradient was observed. Thus, the white medulla was the most active in this respect followed by, in descending order, red medulla and cortex. 14C-Leucine incorporation into protein was completely abolished in the presence of cycloheximide. The distribution of labeled protein between the medium and slice suggests a high degree of cellular integrity and little secretion of labeled protein from slice to medium. The pattern of 14C-leucine incorporation amongst the different zones of kidney of hypophysectomized rats was similar to that noted in normal rats. 相似文献
10.
Axillary buds of Nicotiana tabacum L. cv. Maryland Catterton normally suppressed by apical dominance are released from dormancy with 6-benzylaminopurine. This work was done to determine the change in the [methyl?14C] metabolism from methionine during bud stimulation with cytokinin. Dormant buds metabolize [methyl?14C] -methionine to 14CO2 more effectively than buds released from dormancy. This oxidation of the methyl group is inhibited with benzylaminopurine. On the other hand, the methylation of polar membrane components, including phosphatidylcholine, is enhanced by the cytokinin during the period preceding the increase in bud weight. The interpretation is presented that the enhanced synthesis of membrane components, as well as the preservation of the methyl groups, are mechanisms for the cytokinin release of bud dormancy with 6-benzylaminopurine. 相似文献
11.
S S Raghavan R A Mumford J N Kanfer 《Biochemical and biophysical research communications》1974,58(1):99-106
Radioactive glucocerebroside was formed when purified calf spleen β-glucosidase was incubated in the presence of 4-methylumbelliferyl-β--glucoside and (14C) labeled ceramide. The product was identified by cochromatography on thin layer plates and carrier dilution and crystallization to constant specific activity. The radioactive glucocerebroside was also a substrate for hydrolysis by this same β-glucosidase preparation employed for its synthesis resulting in the liberation of labeled ceramide. Neither methyl-β--glucoside nor free D-glucose were effective in producing radioactive glucocerebroside when incubated with labeled ceramide in this system. 相似文献
12.
The F1-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time () for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F1-ATPase resulted in the incorporation of [14C]DCCD as well as [14C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [14C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [14C]DCCD and 4 mol [14C]NBD-Cl bind per mol F1-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F1-ATPase from M. phlei. 相似文献
13.
Oxidative cleavage of aromatic compounds is often part of a degradative process and is widely observed in nature. The immediate catabolic products can sometimes cyclize or rearrange to new secondary metabolites. The enzymatic contraction of a dehydroisocoumarin to yield cyclopentenoid metabolites in Cryptosporiopsis sp. is reported. The label distribution of (+) cryptosporiopsin, a chlorinated cyclopentenone, was determined by analysis of the [13C]nmr of [1-13C] and [2-13C]acetate enriched-cryptosporiopsin. The putative aromatic precursor of cyclopentenoid metabolites, 2,3-dihydro-6,8-dihydroxy-2-methylisocoumarin (6), was isolated from Aspergillus terreus. This metabolite (6) was prepared doubly labeled (). The aromatic origin of the Cryptosporiopsis chlorinated cyclopentenoid metabolites was rigorously proven from feeding experiments with doubly labeled compound 6. A related but nonchlorinated metabolite, terrein, was isolated from A. terreus and was also shown to be derived from [. 相似文献
14.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(3):342-351
The existence of metabolically distinct pools of in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-14C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-3H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the 3H and 14C label disappearance in during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of . In both yeast and hepatocytes, the ratio in decreased sharply. The individual 3H and 14C decrease in showed values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel 3H and 14C decreases in . 相似文献
15.
The metabolism of 14C-labeled 6-benzylaminopurine in aseptic cultures of Lemna minor was investigated. This cytokinin is slowly taken up by the plants; part of it can be released and part of it is rapidly metabolized to several compounds, among which the corresponding nucleotides can be identified. In this connection the feasibility of locating the site of hormone receptors (sites of primary action) in plants is discussed. Incorporation of the labeled cytokinin into Lemna tRNA was not observed, although tRNA hydrolysates, isolated from plants grown on a cytokinin-free medium, contain a fair amount of cytokinin activity and therefore presumably cy okinin molecules. 相似文献
16.
One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2, 6) - (10) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth , is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?14C] acetate and [methyl-14C] methionine into JH III showing biosynthesis, but no indication of the synthesis of JH I or JH II was seen. 相似文献
17.
S Nasu F D Wicks S Sakakibara R K Gholson 《Biochemical and biophysical research communications》1978,84(4):928-935
Two proteins (A and B) from are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and A protein. In contrast the B protein- A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver- system was due to contamination of commercially available [14C]L-aspartate with [14C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and B protein is L-aspartate oxidase. 相似文献
18.
The light-harvesting chlorophyll -protein complexes (LHCP) of spinach, pea, and barley thylakoids apparently contain four nonidentical polypeptide subunits of between 29,000 and 23,000 daltons on highly resolving sodium dodecyl sulfate-polyacrylamide gradient gels. Trypsin treatment of the spinach complex degraded at least the two major subunits by approximately 2000 daltons and resulted in a three-subunit pattern on gels. Proteoliposomes reconstituted with LHCP and the chloroplast diacyl lipids aggregated markedly in the presence of cations but vesicles containing LHCP prepared from trypsin-treated thylakoids did not. Amino acid analysis of native- and trypsin-treated LHCP indicated that the fragment(s) released by trypsin, which is essential for cation-induced stacking of thylakoids, contains lysine and arginine, but not aspartate or glutamate, and is thus cationic. Carboxyl groups on the surface of LHCP were charge neutralized using a water-soluble carbodiimide (1-ethyl-(3-dimethylaminopropyl)carbodiimide) plus [14C]glycine ethyl ester. Only two or three sites were labeled per 26,000-dalton polypeptide equivalent and only a minor fraction of this (22–24%) was located in the surface fragment(s) released by trypsin. Both LHCP and LHCP proteoliposomes, after carboxyl modification, aggregated avidly at low salt concentrations. The findings suggest that exposed anionic groups on the surface of LHCP contribute to an electrostatic repulsive force between membranes which must be attenuated, either by cation binding or chemical neutralization, before membrane-membrane adhesion can occur. In line with this the binding of Mn2+ by LHCP (approximately four Mn2+ bound/26,000-dalton polypeptide equivalent) was sharply decreased after carboxyl modification. 相似文献
19.
Isolated rat hepatocytes were labeled with [35S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome 1 complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [35S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III. 相似文献
20.
A A Qureshi M Kim F A Lornitzo R A Jenik J W Porter 《Biochemical and biophysical research communications》1975,64(3):836-844
Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [14C]pantetheine-labeled fatty acid synthetase complex [Lornitzo , J. Biol. Chem. , 1654 (1974)]. When [14C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [14C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group. 相似文献