Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.     

Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.     

High density lipoprotein exchange reactions

The Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein. The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively. According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested. The catalytic constants were found to be higher at pH = 7.6 than at pH 10.0 for all the three enzymes. This relative increase was much more pronounced in the case of the porcine enzyme. The KM value at pH = 10.0 was also significantly higher for the porcine enzyme. Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

     

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.     

Storage proteins from seeds of Pinus pinea L

The Mediterranean stone pine Pinus pinea L. (gymnosperm, Pinaceae) is much appreciated for its seed production, widely used in food preparation in the Mediterranean Basin. Seeds contain 25% proteins on a dry-weight basis. Pinus pinea accumulate globulins as major storage proteins in seeds (75% of total storage proteins), composed of several subunits of 10 to 150 kDa, revealed by SDS-PAGE. The albumin fraction (15%) represents three subunits of 14, 24 and 46 kDa. Glutelins, the least soluble fraction, represents a small proportion (10%). Their constitutive units have frequent PM of 43 kDa. Prolamins also represent a very small percentage (1 to 2%).     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes

A bi directional selection experiment was conducted to measure 5-day antibody titers to sheep erythrocytes in White Leghorn chickens. There was an immediate response to selection with significant differences between lines for the selected trait found in the S₁ and all subsequent generations. Comparisons of S₆, S₇ and S₈ generation females revealed differences between lines in disease resistance and in certain reproductive traits such as age at first egg, percentage hen-day egg production, percentage fertility and duration of fertility. The implications of these correlated responses are important to selection programs for general disease resistance.     

Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.     

Allergy to drugs: antioxidant enzymic activities, lipid peroxidation and protein oxidative damage in human blood

Reactive oxygen species lead to lipid peroxidation and specific oxidation of some specific enzymes, proteins and other macromolecules, thus affecting many intra- and intercellular systems. Recently, antioxidant functions have been linked to anti-inflammatory properties. Cell defences against toxic oxygen include antioxidant enzymes. We studied the enzymic antioxidant capacity in human blood of both erythrocytes and mononuclear cells from patients suffering from an allergic reaction to different drugs. We determined superoxide dismutases (SODs), glutathione peroxidase (GSHPx) and catalase (CAT) activities in each cell type. We also determined the extent of thiobarbituric acid reactive substances (TBARS) and the oxidative damage to proteins, in order to study the correlation between the cellular enzymic activities, the oxidative status and the allergic reaction. In mononuclear cells from allergic patients, SODs and CAT activities were enhanced compared with controls. Conversely, a decrease in GSHPx activity was found. In erythrocytes, higher values for CAT, GSHPx and SODs activities were found in allergic patients. TBARS were also enhanced in both types of cells, and the carbonyl content of serum was equally increased. The respective enzymic imbalances in mononuclear cells and erythrocytes, namely, GSHPx/SOD and CAT/SOD, and their consequences are discussed. To our knowledge, this is the first global study of antioxidant enzyme determinations, including TBARS level and carbonyl content, in patients suffering from allergies to drugs.     

Heterogeneous ethoxylation of cellulose: influence of alkali and available-water concentrations on substituent distributions

O-(2-Hydroxyethyl)cellulose (1) as formed by the alkali-catalyzed addition of ethylene oxide to cellulose flock in a slurry process is not uniformly substituted. Most of the ethylene oxide adds to HO-6 in a chain-fashion, so that ～20% of the d-glucose residues remain totally unsubstituted at 2.0 molar substitution. Consequently, an aqueous solution thickened by 1 is highly susceptible to enzymic degradation. Stepwise decrease in concentration of alkali during etherification gives improved stability to enzymic degradation associated with a more-uniform distribution of hydroxyethyl groups between the three hydroxyl groups of the glucose residues in cellulose. The relative reactivities of hydroxyl groups and patterns of substitution were established by matching the distribution patterns from a stochastic computer-model with the distribution of substituents as determined by chemical analyses [namely hydrolysis with sulfuric acid to determine the percentage of unsubstituted glucose residues (u-2) and with periodate oxidation for determining the percentage of unsubstituted 2,3-vicinal diol groups per residue]. The reactivities of the three hydroxyl groups at various alkali concentrations in a heterogeneous, slurry-addition process approximate those observed under homogeneous conditions, wherein the reactivities have been determined by tedious chromatographic analyses. In the variable-alkali procedure for addition of ethylene oxide, the amount of water available to the cellulose matrix in the low-alkali (m) sequence is important both for the stability to enzymic degradation and for obtaining gel-free, thickened, aqueous solutions. Optimal stabilities and gel-free solutions are observed at intermediate water: cellulose ratios of 1.10–1.23. At a ratio of 1.23, the stability to enzymic degradation is less sensitive to percentage variations of u-2 than in 1 prepared at higher or lower water: cellulose ratios. Although the initial degree of degradation between 1 of high molar substitution prepared at 6.8m alkali concentration and a similar product prepared under variable alkali conditions may be related to percentage differences of u-2, the rate and final degree of degradation do not relate to percentage differences of u-2. An adequate interpretation, utilizing known cellulase turnover-rates, is found in stochastic-model projections of the distribution of consecutive 2 residues not substituted at HO-2. The results indicate that (1) more-uniform substitution through equalization of hydroxyl reactivities is achieved by lowering the alkali concentration, and (2) more-uniform substitution of 2 of the many cellulose-chains being substituted is achieved by employing an optimal amount of “available” water during etherification.     

Variation of some serum proteins in red deer, Cervus elaphus L

Various electrophoretic techniques, immunoblotting and inhibitions of trypsin and chymotrypsin were used to study the variability of serum proteins in farmed red deer, Cervus elaphus L., of Czechoslovakian origin. Easily interpretable polymorphisms were observed in transferrin (variants A, B1, B2, C) and vitamin D binding protein, GC (variants D, F, I, S). Great variability was observed in the protease inhibitors PI2, PI3, PI4, PI5, and PI8 and in unidentified zones in the vicinity of albumin, but no genetical or physiological interpretation for this variability is yet available. Haemopexin, alpha 1 glycoprotein, protease inhibitors PI1, PI6 and PI7 were monomorphic.     

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.     

Different proteins in the hemolymph sera from sarcomatous and healthy soft shell clams, Mya arenaria L.

1. Serum proteins showed quantitative and qualitative differences between sarcomatous and healthy soft shell clams, Mya arenaria L. 2. Total protein concentration was significantly higher in the serum of sarcomatous clams than of healthy clams. 3. According to SDS-PAGE, more serum proteins with more variability distinguished sarcomatous clams from healthy ones. 4. Sarcomatous clams had unique serum proteins of M(r)23,000, 45,000 and 54,000. Healthy clams had unique serum proteins of M(r) 24,000, 103,000 and 105,000. 5. During disease progression, sarcoma-specific proteins appeared while normal proteins disappeared. 6. We propose that some sarcoma-associated proteins may have tumor promoting and/or cytotoxic functions and that some normal proteins which disappear during disease progression may be involved in the humoral defense system.     

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

Membrane proteins of human erythrocytes are modified by advanced glycation end products during aging in the circulation

Recent immunological studies demonstrated that proteins in vivo in several diseases are subjected to post-translational modification by advanced glycation end products (AGEs), suggesting a potential role of AGEs in aging and age-enhanced disease processes such as diabetic complications, atherosclerosis and Alzheimer's disease. Nvarepsilon-(Carboxymethyl)lysine (CML) is one of the major AGE-structures demonstrated in vivo so far. In the present study, membrane proteins from young erythrocyte population were compared with those from senescent erythrocytes separated from the same individual in their CML-contents using a monoclonal antibody for CML (6D12). SDS-polyacrylamide gel electrophoresis and subsequent Western blot showed that 6D12 bound to the band 1, 2, 3, 4.2, 5, 6 and 7 proteins from senescent erythrocytes, but not to those from young erythrocytes. Furthermore, quantitative estimation of the reactivity of 6D12 to these erythrocyte membranes by ELISA showed that the reactivity of 6D12 to senescent erythrocyte membranes was 3- to 6-fold higher than that of young erythrocyte membranes. These results indicate that membrane proteins of circulating erythrocytes undergo CML-modification, and the modified proteins accumulated in an age-dependent manner during the life span of erythrocytes.     

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Isolation and characterization of an antigen-specific suppressor inducer molecule from serum of hyperimmune mice by using a monoclonal antibody

We have used a rat monoclonal antibody (mAb) (called 14-30) to affinity purify the antigen-binding chain of a suppressor inducer factor (TsiF-AB) from the serum of mice hyperimmune to heterologous erythrocytes. The TsiF-AB requires the addition of a second, antigen-nonspecific component for biologic activity as well as Lyt-2+ T cells in the assay culture. This mAb can be used to affinity purify suppressor inducer factor from a well-characterized TsiF but not suppressor effector factor (TseF) from culture supernatants. Binding of mAb 14-30 to TsiF is independent of the antigen specificity of the suppressor factor and of the strain of origin of the TsiF. The TsiF affinity purified from hyperimmune serum has an apparent m.w. of 68,000 by SDS-PAGE analysis. 2D gel analysis shows that the serum-derived TsiF has charge heterogeneity, all in the acid range.     

Postfractionation for enhanced proteomic analyses: routine electrophoretic methods increase the resolution of standard 2D-PAGE

Here we have addressed common issues of resolution in two-dimensional polyacrylamide gel electrophoresis (2DE) experiments including proteins 'stacked' at pH extremes, unresolved peptides migrating at the front of separation, and areas of the 2D gel obscured by high abundance proteins. Postfractionation, by selective application of well-established electrophoretic separations immediately following standard 2DE, yields markedly improved resolution in these traditional problem areas using no more specialized equipment or techniques than SDS-PAGE itself.     

A comparison of methods for evaluating seed quality in carrots (Daucus carota)

Comparisons of embryo length, ISTA (International Seed Testing Association), cold (10 ^oC), controlled-deterioration, and slope tests for estimating seedling size variability and percentage seedling emergence were made using eighteen stocks of carrot seeds. The coefficient of variation (C.V.) of seedling weight was closely correlated with the C.V. of embryo length (r = 0–87 D.F., 16) and also with the C.V. of root length from the slope test (r = 0–63 D.F., 16). Poorer estimates of percentage seedling emergence were obtained from the cold test in all three sowings (correlation coefficients were between 0–65 and 0–70 D.F., 16) compared with the ISTA, controlled deterioration, and slope tests. For these three tests the correlation coefficients varied between 0–80 and 0–87 (D.F., 16) for all sowings. The embryo test is a rapid (1 man hour per seed lot) and accurate method of assessing potential variability between seed lots. The slope test could provide a less labour demanding method of assessing potential variability than the embryo length test though the results would not be available immediately. However, the slope test could also provide as good an estimate of seedling emergence in the field as the ISTA or controlled-deterioration tests.