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Ron Milo 《BioEssays : news and reviews in molecular, cellular and developmental biology》2013,35(12):1050-1055
Novel methods such as mass‐spectrometry enable a view of the proteomes of cells in unprecedented detail. Recently, these efforts have culminated in quantitative measurements of the number of copies per cell for most expressed proteins in organisms ranging from bacteria to mammalian cells. Here, we estimate the expected total number of proteins per unit of cell volume using known parameters related to the composition of cells such as the fraction of cell mass that is protein, and the average protein length. Using simple arguments, we estimate a range of 2–4 million proteins per cubic micron (i.e. 1 fL) in bacteria, yeast, and mammalian cells. Interestingly, we find that measured values that are reported for fission yeast and mammalian cells are often about 3–10 times lower. We discuss this apparent discrepancy and how to use the estimate as benchmark to recalibrate proteome‐wide quantitative censuses or to revisit assumptions about cell composition. 相似文献
3.
Isolation and characterization of an extremely basic protein from adenovirus type 5. 总被引:10,自引:9,他引:1
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By starch-gel electrophoresis and a staining method that is highly sensitive for argininyl residues, adenovirus type 5 was found to contain two minor basic polypeptides of extreme cathodic mobility in addition to the two known core proteins. The fastest-migrating polypeptide, named mu protein, and the second fastest polypeptide are found in adenovirions and virus-infected KB cells but not in top components or in uninfected cells. The top components and infected cells contain an additional basic polypeptide, presumably P-VII, that migrates slightly slower than polypeptide VII. None of the basic polypeptides of adenovirions was electrophoretically identical to the host histone. The basic proteins of adenovirions were purified by urea phosphocellulose column chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two minor basic core proteins, mu and another component, have similar mobilities in sodium dodecyl sulfate-polyacrylamide gels as a complex of polypeptides X-XII. After further purification on a Sephadex G-75 column, the mu protein was found to have a molecular weight of about 4,000. Amino acid analysis showed that the mu protein lacks tryptophan and 69% of the total amino acid residues are basic, that is, 54% arginine, 13% histidine, and 2% lysine. Only eight amino acids seem to contribute to make the mu polypeptide. There are 125 copies of the mu polypeptide per 1,000 copies of polypeptide VII in a virion. 相似文献
4.
Varicella-zoster virus p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell. 总被引:4,自引:3,他引:1
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Varicella-zoster virus (VZV) directs the synthesis of numerous glycosylated and nonglycosylated infected-cell-specific proteins, many of which are later incorporated into the virion as structural components. In this study, we characterized a nonglycosylated polypeptide complex with the aid of a VZV-specific murine monoclonal antibody clone, 251D9. As detected by indirect immunofluorescence, the antibody bound mainly to antigens located within the nuclei of infected cells and did not attach to an uninfected cell substrate. The polypeptide specificity of the monoclonal antibody was determined by immunoblot analysis of electrophoretically separated infected cell extracts to react with a 32,000-molecular-weight VZV-specific protein (p32); in addition, the antibody also bound to a 36,000-molecular-weight polypeptide. The synthesis of these antigens was unaffected by inhibitors of glycosylation. Nonionic or ionic detergents were only marginally effective in solubilization of the p32-p36 complex, and relatively small amounts were eluted from nuclei by high salt concentrations (2 M NaCl). The same proteins remained associated with the nuclear matrix of VZV-infected cells. We also demonstrated that the protein complex was a major component of purified VZV nucleocapsids; p32 was especially prominent in both full and empty capsids. Immunoblot analysis of the nucleocapsid preparation revealed two additional species (p34 and p38) in the p32-p36 complex. Phosphorylation was a distinctive feature of some of the constituents. In summary, these results indicate that the p32-p36 complex represents a family of structural proteins closely associated with the assembly of VZV nucleocapsids and the encapsidation of viral DNA. 相似文献
5.
Polypeptides encoded by polyadenylated and non-polyadenylated messenger RNAs from normal and heat shocked HeLa cells 总被引:6,自引:0,他引:6
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There is an increased synthesis of proteins in the molecular weight region of 100,000 72,000-74,000 and 37,000 two hours after treatment of HeLa cells for 10 min at 45 degrees C. In vitro translation, using a rabbit reticulocyte cell-free protein synthesising system, of HeLa cell cytoplasmic RNA shows that the prominent 72,000-74,000 Mr heat shock protein band comprises seven polypeptide species (namely alpha d beta gamma delta epsilon zeta) and these polypeptides are directly encoded by both polyadenylated and nonpolyadenylated mRNA. 相似文献
6.
Three structural polypeptides coded for by minite virus of mice, a parvovirus. 总被引:40,自引:38,他引:2
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Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step. 相似文献
7.
The synthesis of individual proteins in the mouse plasmacytoma cell MPC-11 is differentially inhibited when the rate of polypeptide chain initiation is reduced by exposure of cells to hypertonic medium. The synthesis of immunoglobulin G light and heavy chain polypeptides is 3.5 to 4-fold and 1.5 to 2-fold more resistant, respectively, than the synthesis of non-immunoglobulin G proteins when total protein synthesis is reduced by ~90%. In contrast, when polypeptide chain elongation is inhibited, the synthesis of the light and heavy chains is not more resistant than the synthesis of non-immunoglobulin G proteins.The results with MPC-11 cells suggests that: (1) under standard growth conditions the relative synthesis of individual proteins is determined mainly, but not exclusively, by the relative amounts of the individual messenger RNA species present in the cell; (2) under conditions where the overall rate of polypeptide chain initiation is reduced the relative synthesis of individual proteins becomes more dependent upon the intrinsic ability of their corresponding mRNAs to form functional mRNA-ribosome initiation complexes. 相似文献
8.
Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes.
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Vaults are cytoplasmic ribonucleoprotein structures that display a complex morphology reminiscent of the multiple arches which form cathedral vaults, hence their name. Previous studies on rat liver vaults (Kedersha, N. L., and L. H. Rome. 1986. J. Cell Biol. 103:699-709) have established that their composition is unlike that of any known class of RNA-containing particles in that they contain multiple copies of a unique small RNA and more than 50 copies of a single polypeptide of 104,000 Mr. We now report on the isolation of vaults from numerous species and show that vaults appear to be ubiquitous among eukaryotes, including mammals, amphibians (Rana catesbeiana and Xenopus laevis), avians (Gallus Gallus), and the lower eukaryote Dictyostelium discoideum. Electron microscopy reveals that vaults purified from these diverse species are similar both in their dimensions and morphology. The vaults from these various species are also similar in their polypeptide composition; each being composed of a major polypeptide with an approximate mass of 100 kD and several minor polypeptides with molecular masses similar to those seen in the rat. Antibodies raised against rat vaults recognize the major vault protein of all species including Dictyostelium. Vaults therefore appear to be strongly conserved and broadly distributed, suggesting that their function is essential to eukaryotic cells. 相似文献
9.
Quantitation of the major proteins of the human erythrocyte membrane by amino acid analysis 总被引:2,自引:0,他引:2
The proteins of the human erythrocyte membrane have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resulting gel cut into 2-mm sections, and the amino acid content and composition of each slice measured using a sensitive method of amino acid analysis. The distribution of proteins among bands coincides closely with that estimated using staining intensity. Composition data for the major bands agree well with those reported for the purified proteins in all cases except that of band 4.5. Using quantitative amino acid analysis and resistive particle counting the total protein content of purified membranes was found to be 3.75 X 10(-13) g/cell, which is substantially less than previous estimates based on indirect methods. These data are used to calculate the number of copies of each major protein in a single erythrocyte. 相似文献
10.
To initiate studies on the protein microenvironment of the mouse blastocoel, we examined the electrophoretic profile of newly synthesized proteins secreted into the blastocoel, as well as those secreted into the medium. Although most polypeptides reveal no relative enrichment, some proteins (e.g., proteins of Mr = 155,000 and 33,000) are enriched in the blastocoel relative to those secreted apically into the medium. In addition, some proteins (e.g., proteins of Mr = 102,000 and 40,000) are enriched in the medium relative to the blastocoel. The relative amount of newly synthesized protein secreted into the blastocoel is about 2.5% of total protein synthesis. In addition, the trophectoderm and inner cell mass contribute to these proteins. Results of these studies suggest a potential function for these blastocoel-enriched proteins in inner cell mass development. 相似文献
11.
Identification and preliminary characterization of two related proliferation-associated nuclear phosphoproteins 总被引:9,自引:0,他引:9
S N Malek A I Katumuluwa G R Pasternack 《The Journal of biological chemistry》1990,265(22):13400-13409
Two nuclear phosphoproteins, pp35 and pp32, were purified from A20 cells, a murine B-lymphoblastoid cell line. Initially detected by cross-reactivity with antibodies to human erythrocyte protein 4.1, the 35- and 32-kDa proteins were purified by sequential fractionation of non-ionic detergent cell lysates on DEAE-cellulose, high performance liquid chromatography (HPLC)-anion-exchange chromatography, and HPLC hydroxylapatite chromatography. By two-dimensional peptide mapping, pp35 and pp32 are related but do not appear to represent sequential proteolytic products. Both pp35 and pp32 appear to be associated with cell proliferation. Antibodies specific for pp35 and pp32 show prominent intranuclear staining in A20 cells but only focal staining in normal murine lymphoid tissues. Quantitative immunoblotting showed that both pp35 and pp32 are, respectively, expressed at 5.9 x 10(4) and 7.0 x 10(4) copies/cell in small, dense resting B lymphocytes, increasing approximately 12- and 7-fold after polyclonal stimulation with lipopolysaccharide. When normalized to total cell protein, this represents specific inductions of approximately 4- and 2-fold. Expression of both pp35 and pp32 is constitutively high in populations of neoplastic B cell lines; moreover, both are expressed in the nuclei of intestinal crypt epithelial cells but not in other epithelial compartments in the same sections, suggesting that forms of pp35 and pp32 may be expressed in additional tissues and associated with proliferation. 相似文献
12.
A Vitali L Pacini E Bordi P De Mori L Pucillo B Maras B Botta A Brancaccio B Giardina 《Plant Physiology and Biochemistry》2006,44(10):604-610
A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein. 相似文献
13.
Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis 总被引:18,自引:8,他引:10
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S J Norris N W Charon R G Cook M D Fuentes R J Limberger 《Journal of bacteriology》1988,170(9):4072-4082
The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species. 相似文献
14.
Overexpression of even non-toxic proteins in bacteria causes a starvation-like response: the arrest of bacterial proliferation and apoptotic-like suicidal cell death. We have shown here that, as in the cells of higher organisms, these effects are accompanied by DNA degradation. The fusion with the bacterial MBP of a polypeptide, belonging to the 14-3-3 family and normally expressed in pumpkin (C. pepo), modifies the apoptotic-like effects of overexpression of this protein in E. coli. Fusion of the full length 14-3-3 protein with the MBP considerably slows down the DNA degradation caused by overexpression of the unmodified MBP. Overexpression of the construct containing a truncated version of the 14-3-3 polypeptide causes immediate arrest of bacterial growth and rapid degradation of the chromosomal DNA. This result suggests that the DNA degradation in bacteria is an active process which can be modified to some extent by an endogenous protein. 相似文献
15.
AFP or SA immobilized on nitrocellulose membranes (AFP-NC or SA-NC) were used as affinity matrices to purify cell membrane proteins with affinity for AFP (AFP-BP) and for SA (SA-BP) from membrane-enriched extracts of Raji cells (a B-lymphoma cell line), as well as for normal resting and activated peripheral blood lymphocytes (PBMC). SDS-PAGE and ligand blotting assays showed that AFP-BP and SA-BP isolated from Raji cells are probably identical molecules. They consisted of two sets of polypeptides of 31 kDa and 18 kDa. The glycoprotein nature of isolated 31 kDa and 18 kDa peptides was suggested by positive staining with Schiff's reagent, and amino-acid analysis revealed similar amino-acid composition for the two glycoproteins. In human PHA-activated PBMC, only the 18 kDa polypeptide was identified and isolated as AFP-BP or SA-BP. As in Raji cells, this 18 kDa polypeptide, isolated by affinity for AFP or for SA, appeared to be the same molecule. Contrary to Raji cells and activated PBMC, no proteins with an affinity for AFP or for SA were identified or isolated in resting PBMC. These observations strongly suggest that the isolated 31 kDa and 18 kDa glycoproteins are probably AFP receptors previously demonstrated in several neoplastic and normal cells undergoing growth and/or differentiation; indeed, they were identical to albumin-binding proteins described by others. 相似文献
16.
Pedro M. Sanllorenti Diana B. Tardivo Ruben D. Conde 《Molecular and cellular biochemistry》1992,115(2):117-128
The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a Mr 36 000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The Mr 36 000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers. 相似文献
17.
Jacqueline Mouriquand Claude Mouriquand Elisabeth Petitpas Josette Louis Marie-Ange Mermet 《Biotechnic & histochemistry》1981,56(4):215-219
A modified Papanicolaou staining procedure using diluted Harris' hematoxylin with potassium alum is described. Nucleolar staining varies from blue to bright red. This technique has been applied to mammary tumor cell lines in vitro under several conditions of hormonal stimulation known to induce protein synthesis and cell differentiation. Blue nucleoli are observed in control resting cells, while bright red nucleoli are seen after hormonal stimulation. 相似文献
18.
Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation 总被引:5,自引:0,他引:5
F Mollinedo 《Biochimica et biophysica acta》1986,861(1):33-43
A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation. 相似文献
19.
We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ. 相似文献
20.
Recent studies indicate that the cytoskeleton may be involved in modulating tissue-specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5-1 micrograms/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration-dependent manner, with beta-casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17-31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac-treated cells contained the same amount of poly-A+ RNA, and neither CD nor Ac altered mRNA levels for beta-casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac-treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell-cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue-specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments. 相似文献