2D Polyacrylamide Gel Electrophoresis Research Articles

Fluorescent dual colour 2D-protein gel electrophoresis for rapid detection of differences in protein pattern with standard image analysis software.

The use of two different fluorescent dyes in two-dimensional (2D) polyacrylamide gel electrophoresis was recently described and termed difference gel electrophoresis (DIGE). Thereby differences between protein samples could be accomplished by fluorescently tagging the samples with different dyes as well as co-separation and visualisation in a single gel. We adapted this method to the ampholyte technique, using newly available fluorescent dyes and three common image software systems for analysis. Working with protein lysates from tumour cell lines with defined added proteins we found that the technique is reproducible, sensitive and fast, because it circumvents the necessity of matching several 2D gels. This is mainly due to the fact that the generated images from the two different fluorescent channels could be superimposed by standard image analysis, so that changes in the protein pattern could be easily detected either by a different colour or by comparing grey values of corresponding spots. This method will be especially helpful in comparing proteins from normal and tumour tissue to highlight changes in genesis and progression in cancer.

Characterization of heat-induced aggregates of β-lactoglobulin, α-lactalbumin and bovine serum albumin in a whey protein concentrate environment

Bovine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA), dispersed in ultrafiltration permeate, that had been prepared from whey protein concentrate solution (100 g/kg, pH 6.8), were heated at 75 degrees C. The sequent protein aggregation was studied by one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g beta-lg/kg permeate solution was heated at 75 degrees C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disullphide-bonded dimers, trimers and larger aggregates. When mixtures of beta-lg and alpha-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also served in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of beta-lg. alpha-la and BSA, and 1:1 disulphide-bonded adducts of alpha-la and beta-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heat-treated beta-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of alpha-la. It is likely that when whey protein concentrate is heated under the present eonditions. BSA forms disulphide-bonded strands ahead of beta-lg and that alpha-la aggregation with beta-lg and with itself is catalysed by the heat-induced unfolded BSA and beta-lg.

Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins.

Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion.

Use of two-dimensional gel electrophoresis to measure changes in synovial fluid proteins from patients with rheumatoid arthritis treated with antibody to CD4.

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 microg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.

Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis.

The phosphate starvation response in Bacillus subtilis was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of sigma(B), the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhF confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a sigma(A) promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed with phoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in both phoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by sigma(B)-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a sigma(B)-dependent manner and in the sigB mutant probably via sigma(H). YxiE was induced by phosphate starvation independently of sigma(B) and PhoPR.

Formation of new protein structures in heated mixtures of BSA and alpha-lactalbumin.

The heat-induced protein-protein interactions of alpha-lactalbumin (alpha-La) and bovine serum albumin (BSA), dispersed in a pH 6.8, 10% whey protein concentrates (WPC) permeate, were followed using alkaline and sodium dodecyl sulfate (SDS) 1D and 2D polyacrylamide gel electrophoresis (PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC). Heated (75 degrees C) 5% BSA solution contained large disulfide-bonded BSA aggregates, although some monomer BSA (SDS-monomeric BSA) could be dissociated from the aggregates by SDS. In contrast, similarly heated alpha-La solutions contained small quantities of several monomeric forms of alpha-La and dimeric alpha-La but no large aggregates. When 10% solutions of 1:1 (w/w) mixtures of alpha-La and BSA were heated, large disulfide-bonded aggregates and SDS-monomeric BSA and alpha-La were present. However, heated 2% mixtures contained more modified alpha-La monomers, alpha-La-dimers, and alpha-La-trimers, fewer large disulfide-bonded aggregates, and less SDS-monomeric alpha-La or BSA. These results suggest that BSA forms disulfide-bonded aggregates that contain available thiol groups that can catalyze the formation of differently structured alpha-La monomers, dimers, higher polymers, and adducts of alpha-La with BSA.

Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis.

The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation. Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.

Effect of Heat Treatment on the Conformation and Aggregation of β-Lactoglobulin A, B, and C

Solutions of bovine β-lactoglobulin (β-Lg) A−C were heated at temperatures between 50 and 90 °C for 12.5 min at pH 6.7 or 7.4, and the products were analyzed by alkaline, sodium dodecyl sulfate (SDS), and two-dimensional (2D) (alkaline and then SDS) polyacrylamide gel electrophoresis (PAGE). Results from the pH 6.7 samples that were ∼70% denatured showed that the proportion of β-Lg that was in very large aggregates was β-Lg C > β-Lg B > β-Lg A. 2D PAGE showed that there were a large number of unexpected intermediate products, especially from β-Lg A. These and other results, including the dissociation of disulfide-bonded dimers from trimers and tetramers by SDS, indicate that (1) β-Lg dimers could be important intermediates in the further aggregation of β-Lg, (2) hydrophobically driven associations occur within the aggregates, (3) the mechanism of β-Lg aggregation is not simple, and (4) differences in variant protein behavior are explainable in terms of net negative charge and specific amino acid substitut...

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with M r of approximately 30 000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30 000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography–mass spectrometry, and found to consist of two related species of approximately 28 100 and 26 500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.

TWO-DIMENSIONAL PROTEIN MAPS OFXENOPUSEGGS AND EMBRYOS AT DIFFERENT DEVELOPMENTAL STAGES

Protein expression during the early development ofXenopushas been followed by 2D-polyacrylamide gel electrophoresis (PAGE). The analysis of two-dimensional maps of eggs and embryos at different stages of development has allowed the separation of more than 2000 spots. Identification of numerous polypeptides was obtained in four different ways: (1) immuno-blotting; (2) amino terminal sequence after blotting on to PVDF membranes; (3) comigration; and (4) assignment in comparison with proteins separated by 2D techniques on reference maps such as human liver, red blood cells, plasma and cerebrospinal fluid reported in the Swiss 2D-PAGE Data Base. The maps presented in this report are a step toward the study of the protein expression inXenopuseggs and embryos and may be a powerful working tool sinceXenopusembryos are popular models for the study of development.

Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins.

The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3-10) in the first-dimensional separation. Cells were labelled by [35S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI > 7). The position reproducibility was high and in the range 1-2 mm in the x- and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17 +/- 11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500,000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study.

Characterization of the respiratory chain from cultured Crithidia fasciculata

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplstids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F 0F 1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F 0F 1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F 0 subunits (b and c) and all of the F 1 subunits. The F 0 b subunit has a low degree of similarity to subunit b from other organisms. The F 1 α subunit is extremely small making the β subunit the largest F 1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

8] Two-dimensional polyacrylamide gel electrophoresis

Techniques of polyacrylamide gel electrophoresis (PAGE) are capable of high-resolution separations of proteins, but even under optimal conditions, any of the current one-dimensional methods can resolve only about 100 distinct zones. However, it is often necessary to analyze the samples of a much higher complexity, such as those from whole cells or tissues. This has resulted in the search for electrophoretic methods, with the potential, to separate complex protein mixtures, containing several thousand proteins, and to resolve proteins, sharing similar physicochemical properties. The best approach to this problem is to combine two different one-dimensional electrophoretic procedures into a two-dimensional (2D) technique. The techniques of 2D PAGE described in this chapter now make it possible to separate the reproducibly complex mixtures of proteins, containing several thousand components. The availability of computer systems capable of rigorous qualitative and quantitative analysis of the resulting 2D protein profiles to establish large-scale 2D protein databases, now makes it possible to apply this technology to the study of patterns of protein expression in various areas of biology and biomedicine. Moreover, the development of techniques for the chemical characterization (i.e., amino acid analysis, protein sequencing, mass spectrometry) of proteins separated by gel electrophoresis can be seen, as providing an interface, between studies of gene expression at the protein level, by techniques, such as 2D PAGE and computer databases, with the rapidly expanding body of information, concerning the complexity of gene expression at the DNA level.

Sodium butyrate delays neutrophil apoptosis: role of protein biosynthesis in neutrophil survival.

Sodium butyrate, which directly affects chromatin structure and function in many cells, is as effective as granulocyte-macrophage colony-stimulating factor (GM-CSF) in delaying apoptosis in human neutrophils. Both butyrate and GM-CSF preserved the ability of neutrophils cultured for 22 h in vitro to generate reactive oxidants and express receptors such as CD16. They also delayed apoptotic morphology and DNA fragmentation, and stimulated de novo biosynthesis: newly-labelled polypeptides detected by 2D-polyacrylamide gel electrophoresis after treatment with GM-CSF and butyrate were very similar. Cycloheximide abrogated the effects of both GM-CSF and butyrate. Exposure to butyrate for 1 h did not prime oxidant production and did not up-regulate expression of CD11b. Hence, unlike GM-CSF, butyrate does not stimulate translocation of granules to the plasma membrane. These data suggest that active gene expression is involved in the regulation of neutrophil apoptosis and changes in chromatin structure and function may control apopotosis in these cells.

Two-Dimensional Polyacrylamide Gel Electrophoresis of Proteins Labeled with the Fluorophore Monobromobimane Prior to First-Dimensional Isoelectric Focusing: Imaging of the Fluorescent Protein Spot Patterns Using a Cooled Charge-Coupled Device

A new method for visualizing 2D protein spot patterns is described whereby proteins containing sulfydryl groups are labeled with the fluorophore monobromobimane prior to the isoelectric focusing step of 2D polyacrylamide gel electrophoresis. The method requires the addition of a single reagent and a delay of only 15 min during sample preparation. High resolution spot patterns were obtained without the need for any postelectrophoretic gel handling. The gel patterns were imaged digitally immediately postelectrophoresis while the gels were still within their glass electrophoresis cassettes. The imaging system was based on a cooled charge-coupled device. The fluorescent spot patterns of proteins from the lymphoid cell-line IM9 were similar but not identical to silver-stained gel spot patterns of the same but underivatized sample. There was an overall increase (approx. 10%) in the number of spots visible on the fluorescent gels compared to those visible on the silver-stained gels.

Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase

HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.

Expression of non-MHC-restricted T cell antigen-binding molecules by thymic lymphocytes

Heterologous antisera which recognize non-major histocompatibility complex (MHC)-restricted T cell antigen-binding molecules (TABM) were used to characterize the expression and structure of TABM on thymic lymphocytes. Approximately 70% of thymocytes express membrane molecules bound by anti-TABM antibodies (mTABM). Antibody activity for thymocyte TABM could be removed by adsorption to splenic T cells, but not by adsorption to splenic B cells. Similarly, adsorption of the antiserum to thymocytes or splenic T cells removed antibody activity to a purified TABM whereas adsorption with B cells had no effect. Radioiodinated thymic and splenic T cell mTABM were resolved by 2D-polyacrylamide gel electrophoresis and when reduced, both populations of mTABM migrated primarily as M r, 23,000 proteins with an isoelectric point range of 6.8–7.8. Multimers of this protein were also observed at M r 85–97,000 and 130–150,000 on both thymocytes and splenic T cells. These data indicate that MHC-unrestricted antigen-binding molecules are expressed by a majority of thymocytes and these thymic TABM are structurally and antigenically similar to mTABM on peripheral cells. This suggests an ontogenic relationship between thymic TABM and peripheral TABM.

Protein characterization of sexually mature and immature forms of Schistosoma mansoni

1. 1. Protein composition of different stages of Schistosoma mansoni was compared using specific antisera, 2D polyacrylamide gel electrophoresis and 14-C-leucine incorporation into proteins. 2. 2. Major qualitative differences were detected when an anti-membrane antiserum was used. 3. 3. 2D gel electrophoresis showed that the protein composition varied when mature and immature females were compared, whereas no differences were noted when mature and immature male worms were compared. 4. 4. Experiments measuring protein synthesis by the different schistosome stages confirmed that upon maturation, only the female schistosomes displayed qualitative differences. 5. 5. The protein pattern of the male schistosomes did not vary significantly as a function of development.

Uterine secretory alterations coincident with embryonic mortality in the gilt after exogenous estrogen administration.

Sixteen crossbred gilts were assigned randomly to receive either an i.m. injection of sesame oil (control) or estrogen (E), 5 mg of estradiol valerate, on d 9 and 10 of pregnancy. Gilts were unilaterally hysterectomized on either d 12 and 14 or 16 and 18. Uterine horns were flushed with 20 ml of .9% sterile NaCl solution to recover conceptus tissue. Conceptuses and endometrial explants were cultured for 24 h with 100 microCi [3H] leucine in 15 ml of minimum essential media. After dialysis, culture media were submitted to 2D-polyacrylamide gel electrophoresis and incorporated proteins were analyzed by fluorography. Normal, intact conceptus tissue was recovered from control gilts. Estrogen-treated gilts flushed on d 12 and 14 contained intact conceptuses; however, uteri from two gilts on d 16 and three on d 18 contained degenerating conceptus tissue. Comparison of endometrial polypeptides synthesized in vitro indicated an alteration in E-treated gilts on d 12 through 18. Although similar polypeptides were present, a band of polypeptides with a Mr of approximately 30,000 and pI from 7.9 to 8.9 and a larger, acidic polypeptide (Mr = 100,000, pI 3.5 to 5.0) were faint or absent in E-treated gilts. Conceptuses elongated normally in the altered uterine environment, but failed to survive past d 14 in E-treated gilts. Although loss of specific polypeptides in E-treated gilts coincides with conceptus death, their function in conceptus development or attachment is unknown.

The Effects of Salt Stress on Polypeptides in Membrane Fractions from Barley Roots

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [(35)S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.