Two-dimensional Acrylamide Gel Electrophoresis Research Articles

Alteration of protein profile in cerebral cortex of rats exposed to bisphenol a: a proteomics study

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.

Use of a phosphosensor dye in proteomic analysis of human mutant tau transgenic mice

Recently, we have generated transgenic mice (designated as SJLB) carrying human N279K mutant tau, one of the tau mutations causing parkinsonism linked to chromosome 17 (FTDP-17). SJLB mice mimic some features of behavioral alterations and neuronal pathology of patients with Alzheimer's disease. To investigate how tau dysfunctions cause these features, we examined the expression and phosphorylation levels in SJLB mouse hippocampal proteins using a phosphosensor dye in two-dimensional poly acrylamide gel electrophoresis analysis and mass spectrometry. Calreticulin and tubulin beta4 are significantly more phosphorylated, and heat shock cognate 71 kDa protein, tubulin beta2, vacuolar ATP synthase catalytic subunit A, alpha-internexin, alpha-enolase, ubiquitin carboxyl-terminal hydrolase isozyme L1, and complexin-2 are significantly less phosphorylated in SJLB mice than control mice. These proteins could be new targets for elucidating underlying mechanisms and therapeutic intervention in neurodegenerative diseases.

Identification of a novel p300-specific-associating protein, PRS1 (phosphoribosylpyrophosphate synthetase subunit 1).

CBP [CREB (cAMP-response-element-binding protein)-binding protein] and p300 play critical roles in transcriptional co-activation, cell differentiation, proliferation and apoptosis. Multiple transcription factors associate with CBP/p300. With the exception of the SYT oncoprotein, no proteins have been identified that specifically associate with p300, but not CBP. In the present study, we isolated a novel p300-associated protein for which no interaction with CBP was observed by GST (glutathione S-transferase) pull-down assay using Jurkat cell lysates metabolically labelled with [35S]methionine. This protein bound the KIX (kinase-inducible) domain of p300. Following resolution by two-dimensional acrylamide gel electrophoresis, we identified the KIX-domain-bound protein by MS analysis as PRS1 (phosphoribosylpyrophosphate synthetase subunit 1), a protein essential for nucleoside biosynthesis. This is the first report to demonstrate the existence of a p300 KIX-domain-specific-interacting protein that does not interact with CBP. Thus p300 may play a role in the regulation of DNA synthesis through interactions with PRS1.

Stimulation of the synthesis of ribosomal proteins in regenerating rat liver with special reference to the increase in the amounts of effective mRNAs for ribosomal proteins.

1 Normal and partially hepatectomized rats were pulse labeled for 15 min with [14C]leucine and [3H]leucine respectively. Both kinds of liver homogenates were mixed and the ribosomal protein fraction was purified from the mixed homogenate by chromatographic procedures. The 3H/14C ratio of this fraction was about three times higher than that of total protein, indicating that the synthesis of ribosomal proteins is selectively increased in regenerating rat liver. 2 The free polysomes from normal or regenerating rat livers were incubated with [3H]leucine in the presence of regenerating rat liver supernatant. Ribosomal proteins were then purified, finally by two-dimensional acrylamide gel electrophoresis. Biosyntheses of most of ribosomal proteins by regenerating rat liver polysomes were about 2.5 to 3.6 times higher than those by normal rat liver polysomes. 3 Using a wheat germ cell-free system, the template activities of poly(A)-containing mRNAs for ribosomal proteins were compared between normal and regenerating rat livers. The template activities at 6, 12, 18, 24 and 48 h after partial hepatectomy were about 1.3, 1.8, 2.4, 1.5 and 1.3 times higher, respectively, than the activity of normal rat liver. The extents of increase in the template activity of poly(A)-containing mRNAs for most of individual ribosomal proteins separated by two-dimensional gel electrophoresis were not so different at 18 h after the operation and the average template activity was 2.8 times higher than that of normal rat liver. The enhanced activity of regenerating rat liver for ribosomal protein synthesis is mainly caused by the increase in the apparent concentrations of mRNAs for ribosomal proteins.

Ribosomal proteins cross-linked to 28-S and 18-S rRNA separated by sedimentation after ultraviolet irradiation of rat-liver ribosomes.

Rat liver 80-S ribosomes, irradiated with 254 nm ultraviolet light for 15-60 min (0.63-2.5 X 10(5) quanta/ribosome), were treated with sodium dodecyl sulfate; 18-S and 28-S RNA fractions were isolated by sucrose density-gradient centrifugation. The protein moieties of the 18-S and 28-S rRNA fractions were labeled with 125I. After digestion of the rRNA fractions with RNase A and T1, the cross-linked proteins were analyzed by two-dimensional acrylamide gel electrophoresis and then autoradiography. Cross-linked proteins were examined further by dodecyl sulfate/acrylamide gel electrophoresis of individual radioactive spots of proteins and their proteolytic peptides on two-dimensional gels. The results showed that S2 and S15 were cross-linked to 18-S rRNA, and L5, L6 and L8 to 28-S rRNA. Similar results were obtained when isolated 40-S or 60-S subunits were irradiated.

Effects of low dose actinomycin D treatment in vivo on the biosynthesis of ribosomal proteins in rat liver.

The long-term effects (up to 12 h) of low dose in vivo actinomycin D treatment, which selectively inhibits rRNA synthesis, on the activity of rat liver for the synthesis of ribosomal proteins relative to that for the synthesis of total protein were investigated. The effects of actinomycin D treatment in vivo and in vitro on the template activity of poly(A)-containing mRNA of rat liver for ribosomal proteins were examined by using a wheat germ cell-free system. The following results were obtained. 1. The activity of rat liver for synthesizing total protein observed in vivo and in vitro was inhibited by actinomycin D treatment even at a small dose. 2. A double-labeling technique using [3H] and [14C]leucine in vivo showed that the rate of synthesis of the ribosomal protein fraction relative to that of total protein in actinomycin-treated rat liver (6 + 6 h) was 1.45 times higher than that in the control rat. 3. By using a wheat germ cell-free system, it was shown that the template activity of poly(A)-containing mRNA for the synthesis of total protein was increased slightly by actinomycin D treatment in vivo. Furthermore, the template activity for the ribosomal protein fraction relative to that for total protein was increased. This increase was observed in most of the ribosomal proteins separated on two-dimensional acrylamide gel electrophoresis, although the extents of increase were different among individual ribosomal proteins examined. On the other hand, the selective increase of the template activity for the ribosomal protein fraction was not observed when poly(A)-containing mRNA was incubated with actinomycin D in vitro, although the template activity for total protein was increased slightly.

Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation

After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultravioletirradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein; these proteins are designated according to the proposed uniform nomenclature. The correlation between that and our nomenclature was reported by McConkey et al. (1979) Mol. Gen. Genet. 169, 1–6. They also confirm the results previously reported (Terao, K., Takahashi, Y. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 230–237).

TRNA's and priming of RNA-directed DNA synthesis in mouse mammary tumor virus.

The low-molecular-weight RNAs of mouse mammary tumor virus (MuMTV) were examined by two-dimensional acrylamide gel electrophoresis. Unlike other retroviruses, MuMTV was found to contain only two major fractions of tRNA. These have been fully characterized and shown to correspond to the published sequences for tRNA1+2Lys and tRNA3Lys. By determining which of these tRNA's was most tightly associated with the MuMTV genome and which of them acquired label from [alpha-32P]deoxynucleoside triphosphates in limited DNA synthesis reactions, we identified tRNA3Lys as the primer for MuMTV reverse transcription in vitro. tRNA3Lys does not share any unusual sequence feature with the other previously characterized retrovirus primers, tRNATrp and tRNAPro.

Identification of neighbouring protein pairs in the rat liver 40-s ribosomal subunits cross-linked with dimethyl suberimidate

1. (1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. 2. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. 3. (3) The following dimers were proposed from these results: S3-S12 (S3 or S3a-S11), S4-S12 (S3b-S11), S5-S7 (S4-S6), S5-S22 (S4-S23 or S24), S6-S8 (S5-S7), S8-S16 (S7-S18), S17-S21 (S16-S19) and S22A-S22B (S23-S24), designated according to our numbering system [1]. The designations according to the proposed uniform nomenclature [2] are described in parenthesis.

Analysis of proteins of the stria vascularis of the normal and the waltzing guinea pig.

Proteins of the stria vascularis of the normal and the genetically deaf waltzing guinea pig were analysed by one and two-dimensional acrylamide gel electrophoresis. Cochlear proteins were labeled in vivo by replacing the perilymph with a solution containing radioactive precursors. With the two-dimensional analysis, more than 200 polypeptides were resolved. Proteins that are exposed on the endolymphatic surface of the stria vascularis were identified by lactoperoxidase-catalysed iodination. Seven polypeptides were identified with this technique. No consistent changes in protein patterns of the stria vascularis from the waltzing guinea pig were detected.

Immunological and electrophoretic studies of human tear proteins

The proteins in tears have been demonstrated to be highly heterogeneous; at least 60 components can be counted in a two-dimensional acrylamide gel electrophoresis. Monospecific antisera and tear proteins antisera were used for the study of these proteins. With this method, thirteen proteins were identified, seven of them behaving identically in tears and in serum. Use of human tear antiserum adsorbed eith human serum demonstrated that some of the components were tear specific. A novel crossed immunoelectrophoretic system was developed to demonstrate that specific tear prealbumin is composed of at least two immunological components. These results confirm and emphasize the complexity and the distinctiveness of human tear proteins.

Proteins of Small Subunits of Rat Liver Ribosomes that Interact with Poly(U)

(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.

Studies on the protein components of 110S and total ribonucleoprotein particles of rat liver nucleoli.

Proteins of rat liver nucleolar RNP, especially 110S RNP containing 45S RNA, were investigated and the following results were obtained. 1. Nucleolar extract was prepared from thioacetamide-treated rat liver nucleoli in the presence of 1 mg PVS per ml. Sucrose-density gradient centrifugation of the nucleolar extract showed RNP distributed between 60S and 110S. The 110S particles (110S RNP) contain 45S RNA as judged from the radioactivity profiles of nucleolar extract labelled in vivo with [3H]orotic acid for 10 min and 2 h. The protein components of 110S RNP and total RNP heavier than 60S (total RNP) were analyzed and compared. 2. Since the effects of PVS treatment on the extraction and the electrophoretic pattern of ribosomal proteins were removed by increasing the Mg2+ concentration during acetic acid extraction, as described in our preceding paper (1), proteins of 110S RNP as well as total RNP from PVS-pretreated nucleoli were extracted with 67% acetic acid containing 334 mM Mg2+ and analyzed by two-dimensional acrylamide gel electrophoresis. Ribosomal proteins masked by contaminating histone spots on the gel were identified by SDS-acrylamide gel electrophoresis. 3. 110S RNP contained a large portion of ribosomal proteins from large subunits; 24 protein spots were distinct, 6 protein spots were faint and 5 proteins (L3, L8, L30, L35, and L36) were missing completely. On the other hand, 110S RNP contained a small number of small subunit proteins; only 6 proteins showed distinct spots, 7 proteins showed faint spots and 12 protein spots were missing. 110S particles contained 11 non-ribosomal proteins, although it is difficult to rule out the possibility that some of them were contaminating chromatin proteins. 4. Total RNP showed electrophoretic patterns of 60S ribosomal proteins similar to those of 110S RNP, although L3 protein was present as a faint spot and 26 proteins showed distinct spots. Total RNP contained more small subunit proteins than 110S RNP; 7 proteins spots were distinct, 10 protein spots were faint and 8 protein spots were missing. The results suggest that some kinds of 40S proteins and L3 protein are attached to 110S RNP during the processing of 110S RNP.

Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis.

We describe a method for examining the state of phosphorylation of initiation factor eIF-2 in reticulocyte lysates. The procedure involves incubation of the lysate with iodo[1-14C]acetate in 8.5 M urea, and fractionation of the labelled proteins by two-dimensional acrylamide gel electrophoresis. This approach has been used to show that haem deficiency, double-stranded RNA, and oxidised glutathione, which all inhibit the initiation of protein synthesis in an analogous manner, all cause a net increase in the level of phosphorylated eIF-2 in the complete lysate protein synthesis system.

In situ cross-linking of vesicular stomatitis virus proteins with reversible agents

Several reversible cross-linking agents were tested for their ability to cross-link the proteins of intact vesicular stomatitis virus (VSV). Results were analyzed by two-dimensional acrylamide gel electrophoresis in the sodium dodecyl sulfate-discontinuous buffer system. Formaldehyde cross-linking was found to be reversible by heat, and gave results similar to the reagent dimethyl-3,3′-dithio-bisproprionimidate (DTBP), cleavable by reduction of its internal disulfide bond. These two reagents caused the formation of cross-linked polypeptides with molecular weights consistent with the species N:M, G:M, and G:N, where N, M, and G stand for the VSV nucleoprotein, membrane protein, and glycoprotein, respectively. Other agents tested included o-phenanthroline in the presence of Cu 2+ and O 2, and the heavy metal ions Cu 2+ and Ni 2+, all reversible by β-mercaptoethanol. These agents gave similar results: No cross-linking of G proteins was observed, and of the possible heterodimers, only N:M was found. NS, the phosphopolypeptide of VSV, was found in complexes corresponding in molecular weight to homodimer and homotetramer species, possibly indicating a tetrameric configuration of the native NS protein. The apparent molecular weights of cross-linked polypeptides were found to be about 20% greater than for linear polypeptides of the same molecular weight, when the mobility in acrylamide gels of a number of cross-linked species from native proteins, enzymes, and viral nucleocapsids was compared to the mobility of linear polypeptides. The accessibility of sulfhydryl groups in native and SDS-disupted VSV was assayed by reaction with radioactive iodoacetic acid; it was found that the G protein contained no free sulfhydryl groups, explaining its failure to react with o-phenanthroline/Cu 2+ and heavy metal ions. Removal of the G proteins from the virus with nonionic detergent prior to cross-linking with DTBP showed the coordinate loss of both the G and the N spots corresponding in apparent molecular weight to the G:N heterodimer. This result provides evidence that the glycoprotein of VSV may be capable of interacting in some manner with the core nucleoprotein.

Identification of enzymically inactive apocytochrome c peroxidase in anaerobically grown Saccharomyces cerevisiae.

Anaerobically grown yeast cells lack cytochrome c peroxidase activity but rapidly acquire it upon aeration. In order to study the oxygen-induced formation of this hemoprotein, extracts of anaerobic and aerobic yeast cells were resolved by one- and two-dimensional acrylamide gel electrophoresis and the separated polypeptides were then checked for comigration with radiolabeled purified cytochrome c peroxidase from aerobic cells or for reaction with cytochrome c peroxidase antiserum. Both types of extracts contained roughly equal amounts of a polypeptide which was indistinguishable from apocytochrome c peroxidase with respect to antigenicity, isoelectric point, and apparent molecular weight in three different gel systems. In confirmation of an earlier report by Sels. A.A., and Cocriamont, C. (1968) (Biochem. Biophus. Res. Commun. 32, 192-198) the oxygen-induced formation of cytochrome c peroxidase was insensitive to inhibitors of protein synthesis and could be mimicked by the addition of heme to extracts of anaerobic cells. We conclude that the oxygen-induced formation of yeast cytochrome c peroxidase involves the addition of heme to the apoenzyme which is already present in the anaerobically grown cells.

Analysis of H-2 and Ia molecules by two-dimensional gel electrophoresis.

Mouse lymphocyte H-2 and Ia glycoproteins have been analyzed with a two-dimensional (2-D) acrylamide gel electrophoresis technique, in which proteins are separated first according to their charge in isoelectrofocusing gels and then according to their size in sodium dodecyl sulfate gels. Individual polypeptide chains from radiolabeled cells are resolved as discrete spots on autoradiograms of the gels, forming patterns which are characteristic of the proteins in the sample. 2-D gels of H-2K, H-2D, and Ia glycoproteins immunoprecipitated from 35S-methionine-labeled cells reveal that these proteins exist in the cells as complex arrays of molecules heterogeneous in both size and charge. Lactoperoxidase-catalyzed radioiodination of lymphocyte surfaces labels only subsets of the total H-2 and Ia molecules with 125I, indicating that some of the molecules may represent cytoplasmic precursors of the cell surface proteins. This theory is supported by the kinetics of labeling of various spots in 35S-methionine pulse-chase experiments. The 2-D gel patterns obtained for both H-2 and Ia antigens have also been shown to be haplotype-specific and independent of the genetic background.

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast.

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized. It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 X 10(-6)M and 5.5 X 10(-5)M, respectively. Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [gamma-32P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.

Synthesis of Ribosomal Structural Proteins by Postmitochondrial Supernatant from Regenerating Rat Liver1

1. When the postmitochondrial supernatant (PM-supernatant) from regenerating rat liver was incubated with [3H]methionine, the incorporation of [3H]methinoine into the N-terminal residues of nascent peptides on ribosomes was observed. This incorporation was sensitive to a low cocentration (2X10(-6) M) of pactamycin. The results suggest that PM-supernatant has low but definite activity for the initiation of nascent protein synthesis. Polysomes and cell sap from regenerating rat liver showed negligible pactamycin-sensitive incorporation of [3H]methionine into N-terminal, residues of nascent peptides. 2. PM-supernatant from regenerating rat liver was incubated with [35S]methionine in the complete reaction mixture. After addition of ribosomal proteins labelled with [3H]methionine in vivo, ribosomal structural proteins were prepared from the incubation mixture by acetic acid extraction, CM-cellulose column chromatography, Sephadex G-200 gel filtration and finally by two-dimensional acrylamide gel electrophoresis. Incorporation was observed in the greater part of ribosomal proteins on the two dimensional gel. From the 35S-to-3H ratios of ribsomal protein fractions during the purification procedures, it appeared that the incorporation of labelled methionine into the ribosomal proteins by PM-supernatant was about 3% of that into the total proteins. When [3H]leucine was used, the values were about 4% in the same cell-free system and 5 to 6% in in vivo labelling. The results indicate that ribosomal proteins are synthesized with high efficiency by PM-supernatant from regenerating rat liver.

Study of mammalian ribosomal protein reactivity in situ: II. — Effect of glutaraldehyde and salts

Results concerning ribosomal protein sensitivity to glutaraldehyde were compared to protein depletion studies using LiCl centrifugation. The relative degree of reactivity of the different proteins was determined by two-dimensional acrylamide gel electrophoresis, and the activity of the reacted subunits was measured. The results obtained mostly confirmed the studies of methoxynitrotropone reactivity reported earlier. For example, L16, L25, L29, L30, L31, S18, S20 appeared to be definitely exposed to both NH2-reagents and LiCl. Some interesting points emerged from this study regarding protein topography in both subunits: (1) with few exceptions, almost all ribosomal proteins were accessible to the surrounding medium; (2) the sensitivity of the 40S proteins to the three reagents used was lower than was that of the 60S proteins; (3) the reactivities of the subunit components changed when subunits were associated: L8 was more reactive with glutaraldehyde in 60S subunits than in 80S ribosomes. In contrast, S14, S15 and S19 were more exposed in ribosomes than in the 40S subunits.