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1.
P J Anderson 《The Biochemical journal》1979,179(2):425-430
Double-labelling and peptide isolation have been used to examine the homology between the actin of IMR-90 human embryo fibroblasts and muscle actin. After separation of mixtures of [14C]carboxymethylated muscle actin and [3H]carboxymethylated proteins of IMR-90 cells of electrophoresis on sodium dodecyl sulphate-containing polyacrylamide gels, peptides were generated from the material co-migrating with actin by digestion with chymotrypsin. Peptides homologous with peptides accounting for Cys-217, Cys-256, Cys-284 and Cys-373 of muscle actin are present in this material, but no peptide homologous with a Cys-10-containing peptide was detected. From the amount of actin-derived peptides present, the actin content of IMR-90 fibroblasts was calculated to be 4.2% of the total protein of these cells. 相似文献
2.
Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine 总被引:2,自引:0,他引:2
A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin. 相似文献
3.
An 18000-dalton protein metabolically labeled by polyamines in various mammalian cell lines 总被引:1,自引:0,他引:1
K Y Chen 《Biochimica et biophysica acta》1983,756(3):395-402
The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [14C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under such incubation conditions, a single protein band with an apparent molecular weight of 18000 was radioactively labeled. [14C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of gamma-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [14C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation. 相似文献
4.
Toxin I from Anemonia sulcata, a major component of the sea anemone venom, consists of 46 amino acid residues which are linked by three disulfide bridges. The [14C]carboxymethylated polypeptide was sequenced to position 29 by automated Edman degradation. The remaining sequence was determined from cyanogen bromide peptides and from tryptic peptides of the citraconylated [14C]carboxymethylated toxin. Toxin I is homologous to toxin II from Anemonia sulcata and to anthopleurin A, a toxin from the sea anemone Anthopleura xanthogrammica. These toxins constitute a new class of polypeptide toxins. No significant homologies exist with toxin III from Anemonia sulcata nor with known sequences of neurotoxins or cardiotoxins of various origin. 相似文献
5.
The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit. 相似文献
6.
Kuang Yu Chen 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,756(3):395-402
The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [14C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [14C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [14C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation. 相似文献
7.
The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA. 相似文献
8.
The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.
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1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase. 相似文献
9.
A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
The conversion of [4 14C]corticosterone[( 14C]B) and 11-deoxy-[1,2-3H]corticosterone [( 3H]DOC) to steroidal carboxylic acids was studied in the BALB/c mouse. There was rapid and preferential excretion of [3H]DOC metabolites into the gastrointestinal tract. Excretion of 14C through the kidney was higher than 3H excretion. Within minutes of intraperitoneal injection, levels of 3H and 14C in most organs reached their maximal levels and subsequently decreased in an exponential pattern. The majority of the organs took up 14C to a greater extent than 3H. Using tissue blood ratio of tracer (T/B) as criterion, it was found that liver, gall bladder, intestine, and kidney concentrated 3H and 14C-labeled steroid from blood. T/B for 3H exceeded that for 14C in the gastrointestinal tract. Abdominal fat preferentially took up [3H]DOC tracer, whereas [14C]B tracer was not taken up by this tissue. T/B was less than 1 for 3H and 14C in heart, thymus, spleen, brain, skeletal muscle and skin. In these organs uptake of B and its metabolites was greater than that of DOC and its metabolites. In liver, [14C]B and [3H]DOC were converted to carboxylic acid metabolites which accumulated in the intestine. The most abundant acid was 11 beta,20 alpha-dihydroxy-3-oxo-pregn-4-en-21-oic acid from B. The acid metabolites of DOC were not identified. For both steroids, acids were major metabolic end-products. 相似文献
11.
Different reactivities of brain and erythrocyte tubulins toward a sulfhydryl group-directed reagent that inhibits microtubule assembly 总被引:2,自引:0,他引:2
R F Ludue?a M C Roach M A Jordan D B Murphy 《The Journal of biological chemistry》1985,260(2):1257-1264
There is considerable evidence that tubulin exists in multiple isotypes, differing in amino acid sequence and tissue distribution. Little is known, however, about the functional significance of these isotypes. Chicken erythrocyte beta-tubulin has been shown by peptide mapping to differ significantly from chicken brain beta-tubulin (Murphy, D. B., and Wallis, K. T. (1983) J. Biol. Chem. 258, 7870-7875). We now find that when the two tubulins, in their native states, are incubated with N,N'-ethylenebis(iodoacetamide) (EBI), a bifunctional sulfhydryl-directed reagent, microtubule assembly by brain tubulin is much more sensitive to inhibition by EBI than is erythrocyte tubulin assembly. The resistance of erythrocyte microtubule assembly to inhibition by EBI is correlated with a low reactivity of erythrocyte tubulin with [14C]EBI. This difference is most marked in the beta subunit which reacts 15 and 17% as well, respectively, with [14C]EBI as do the beta 1 and beta 2 subunits of brain tubulin. Also, erythrocyte beta reacts about 33% as well as does brain beta with iodo[14C]acetamide. These results suggest that a reactive sulfhydryl group, whose oxidation prevents microtubule assembly, is present in brain tubulin but absent or inaccessible in erythrocyte tubulin. Since purified erythrocyte tubulin self-aggregates much more readily than does brain tubulin, it is conceivable that erythrocyte and brain tubulin may differ in that the latter may have its assembly subject to a complex regulation, while erythrocyte tubulin assembly may be regulated by a simpler mechanism. 相似文献
12.
Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state. 相似文献
13.
John Bridgen 《The Biochemical journal》1971,123(4):591-600
Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-(14)C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from rabbit actin. One of the radioactive peptides was the C-terminal peptide and another sequence probably contained the cysteine residue from the N-terminal region of the protein. 相似文献
14.
In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium 总被引:7,自引:0,他引:7
G M Langford 《Experimental cell research》1978,111(1):139-151
It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [3H]serine and [14C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell. 相似文献
15.
Brain tubulin was labeled in vitro by post-translational incorporation of [14C]-tyrosine or in vivo by intra-cranial injection of [3H]-leucine. The labeled protein was purified by ion-exchange chromatography. After incubating at 37 degrees C with a microsomal membrane preparation from rat brain, part of the labeled soluble tubulin became sedimentable at high-speed centrifugation. This was independent of the native configuration of tubulin, the state of tyrosination of the COOH-terminus, or the presence of 100 microM colchicine in the mixture. In addition, the double-labeled tubulin-colchicine complex obtained from the binding of [3H]-colchicine to [14C]-tyrosinated tubulin, bound to the membrane preparation to the same extent as [14C]-tyrosinated tubulin. The data show that either tubulin or the complex resulting from its binding to colchicine distributed between the soluble and the membrane fractions when mixed at 37 degrees C with a microsome preparation. Seemingly, the site for colchicine binding to tubulin needs not to be free for the protein-membrane association. 相似文献
16.
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
17.
AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS 总被引:7,自引:6,他引:1
Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-14C]glucose and [3H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [3H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of 3H to 14C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain. 相似文献
18.
It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila Kc cells. [3H]Mevalonate was incorporated into 54 Kc cell peptides (18-92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C18 radio-HPLC tryptic digest profiles for delipidized, [3H]mevalonate-labeled (a) insect (Drosophila and Spodoptera frugiperda) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) Kc cell nuclear lamin, and (c) a 23.5-kDa purified Kc cell GTP-binding protein were compared and analyzed. [35S]Cysteine-labeled Kc cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [3H]mevalonate-labeled cells. A significant fraction (30-33%) of the doubly labeled tryptic peptides were eluted with greater than or equal to 93% acetonitrile. Kc cell nuclear lamin tryptic digests yielded a single 3H-labeled product which migrated as S-farnesylcysteine. The Kc cell 23.5-kDa GTP-binding protein's 3H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with greater than or equal to 93% acetonitrile. Moreover, the 3H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [3H]mevalonate-labeled Kc and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics. 相似文献
19.
Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material 总被引:13,自引:6,他引:13
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The preparation and purification of cyanogen bromide fragments from [14C]carboxymethylated coelacanth triose phosphate isomerase is presented. The automated sequencing of these fragments, the lysine-blocked tryptic peptides derived from them, and also of the intact protein, is described. Combination with results from manual sequence analysis has given the 247-residue amino acid sequence of coelacanth triose phosphate isomerase in 4 months, by using 100mg of enzyme. (Two small adjacent peptides were placed by homology with the rabbit enzyme.) Comparison of this sequence with that of the rabbit muscle enzyme shows that 207 (84%) of the residues are identical. This slow rate of evolutionary change (corresponding to two amino acid substitutions per 100 residues per 100 million years) is similar to that found for glyceraldehyde 3-phosphate dehydrogenase. The reliability of sequence information obtained by automated methods is discussed. 相似文献
20.
Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry 总被引:2,自引:0,他引:2
Gruhler A Schulze WX Matthiesen R Mann M Jensen ON 《Molecular & cellular proteomics : MCP》2005,4(11):1697-1709
Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling. 相似文献