Electrophoretic analysis of snake venoms

Electrophoretic analyses were conducted on snake venoms from 21 species representing Elapidae, Crotalidae and Viperidae. Denatured and native venoms were analyzed by polyacrylamide gel electrophoretic (PAGE) methods with sodium dodecyl sulfate (SDS) and without SDS. Both SDS-PAGE and PAGE profiles of venoms from different snake species indicate that some proteins and polypeptide components of these venoms have common electrophoretic characteristics suggesting a genetic relationship. Conversely, the electropherograms also showed the characteristic protein and polypeptide profiles that could differentiate one snake species from another. Therefore, both SDS-PAGE and PAGE profiles suggest that proteins and polypeptides with similar characteristics abound among subspecies or related species, although each venom has a unique profile that differentiates one species from the other.     

A Comparison of Spore Polypeptides in Microsporidia from a Grasshopper Control Program1

Spore polypeptide profiles of Nosema locustae and an unidentified microsporidium infecting Aulocara elliotti and Psoloessa delicatula are compared with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Unique spore polypeptide profiles are not detected for the unknown microsporidium from A. elliotti and P. delicatula whereas these profiles are distinctly different from N. locustae spore polypeptide profiles. These results indicate that the microsporidium is not a polymorphic form of N. locustae but a separate species.     

Proteomic profiles of thylakoid membranes and changes in response to iron deficiency

The proteomic profile of thylakoid membranes and the changes induced in that proteome by iron deficiency have been studied by using thylakoid preparations from Beta vulgaris plants grown in hydroponics. Two different 2-D electrophoresis approaches have been used to study these proteomes: isoelectrical focusing followed by SDS PAGE (IEF-SDS PAGE) and blue-native polyacrylamide gel electrophoresis followed by SDS PAGE (BN-SDS PAGE). These techniques resolved approximately 110–140 and 40 polypeptides, respectively. Iron deficiency induced significant changes in the thylakoid sugar beet proteome profiles: the relative amounts of electron transfer protein complexes were reduced, whereas those of proteins participating in leaf carbon fixation-linked reactions were increased. A set of polypeptides, which includes several enzymes related to metabolism, was detected in thylakoid preparations from Fe-deficient Beta vulgaris leaves by using BN-SDS PAGE, suggesting that they may be associated with these thylakoids in vivo. The BN-SDS PAGE technique has been proven to be a better method than IEF-SDS PAGE to resolve highly hydrophobic integral membrane proteins from thylakoid preparations, allowing for the identification of complexes and determination of their polypeptidic components.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

Serological differentiation of microsporidia with special reference to Trachipleistophora hominis

Myositis is a common clinical syndrome in advanced stages of AIDS. Trachipleistophora hominis (phylum Microspora) has been detected in several cases of painful, immobilising myositis in AIDS patients. Enzyme linked immunosorbent assays (ELISAs) and Western blotting of protein profiles separated by SDS PAGE were used to determine whether this species could be detected and differentiated by serology. Sixteen microsporidia, including several species known to infect man and species infecting fish, crustaceans and a mosquito, were used as antigen. Each species had a unique profile of SDS PAGE-separated proteins. In Western blots, mouse antiserum, raised to T. hominis and selected for its high ELISA specificity, bound to antigens ranging from less than 25 kDa to greater than 250 kDa with major bands at 39-44 kDa and 98-150 kDa on T. hominis protein profiles. The serum also recognised some high molecular weight antigens in the profiles of Vavraia culicis, Heterosporis anguillarum, and three species of Pleistophora but none in the remaining genera examined. It was concluded that ELISA and Western blotting could be used to detect and differentiate T. hominis in muscle biopsy tissue from patients with myositis. However, sera from T. hominis-infected patients in the terminal stages of AIDS would not be useful for detection of infections because of a sharp decline in antibody level.     

Rapid identification of medically important yeasts by electrophoretic protein patterns

A simple electrophoretic method for yeast identification was evaluated. Whole cells were extracted by SDS and the protein profiles obtained in SDS-PAGE after Coomassie blue staining were compared for 52 strains from 9 species of yeast or yeast-like fungi commonly isolated from man (Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. tropicalis, Geotrichum candidum, Saccharomyces cerevisiae). The corresponding patterns showed 30 to 45 polypeptides in the range 95-20 kDa and were clearly different for the 9 species. No differences could be detected between strains from the same species. The characteristic patterns were obtained within 24 h allowing rapid identification of the most commonly encountered clinical yeast isolates.     

Immunological studies with the HA1 and HA2 polypeptides of influenza A virus haemagglutinin.

HA1 and HA2 polypeptides of influenza A virus haemagglutinin (HA) were separated in purified form using electrophoresis in SDS containing polyacrylamide gels (PAGE) or chloroform-methanol extraction. The populations of HA1 polypeptides were immunogenic but considerably less so than the intact HA molecule and induced antibody which cross-reacted with influenza A and B viruses. After absorption with heterologous influenza B virus, the cross-reacting antibodies were removed and the HA1 antisera then possessed antibodies which reacted only with the cross-reactive (CR) determinants of the HA of the homologous influenza A virus and viruses of the same subtype. Neither strain-specific (SS) nor virus-neutralizing antibodies were detected in these anti-HA1 sera. HA2 polypeptides were less immunogenic and anti-HA2 antisera after absorption with influenza B virus failed to react with influenza A virus in immuno double diffusion tests and only reacted with partially denatured HA in the more sensitive single radial diffusion tests.     

Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age

Salivary gland proteins were studied in sandflies (Diptera: Psychodidae: Phlebotominae) by electrophoretic techniques. In Phlebotomus duboscqi Neveu-Lemaire the protein concentration was about 30 times higher in females than in males. SDS PAGE revealed eight major bands of 29-62 kDa in salivary gland extracts (SGE) from females, whereas only one band of 57kDa was detected in males. The number of protein components in SGE gradually increased with the age of females. In P. papatasi (Scopoli) the typical electrophoretic pattern was reached in 3-5 days after imago emergence, depending on the temperature at which females were maintained. All major protein components of the female SGE were present in the content of glands. Female SGE were compared in seven colonies of five Phlebotomus and Lutzomyia species; electrophoretic profiles distinguished between species and even between colonies of different geographical origin. In general, the highest variability of major protein components was observed in the 38-48kDa region. Four colonies of the subgenus Phlebotomus (P. duboscqi and P. papatasi) possessed common mobility polypeptides, the highest similarity was found between two colonies of P. papatasi. Other species tested significantly differed, specific prominent bands of 33, 35 and 38kDa were found in P. halepensis Theodor, P. perniciosus Newstead and Lutzomyia longipalpis (Lutz & Neiva), respectively. Glycoproteins in SGE of Lu. longipalpis and P. duboscqi females were identified and analysed using blotting with five lectin conjugates. Specific reaction of lectins ConA and WGA revealed the complex type of N-glycans in the 48 and 53-54kDa glycoproteins present in both species. Similar glycosylation was detected in species-specific bands of the 57-60 and 65-67 kDa in P. duboscqi and Lu. longipalpis, respectively. The high mannose type of glycosylation was found in the 20 and 39 kDa polypeptides of Lu. longipalpis and the 40-42 kDa polypeptides of P. duboscqi. Innate lectin activity specific for aminosugars was detected in SGE of P. duboscqi females using haemagglutination tests with rabbit erythrocytes.     

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.     

Conformational change of the haemolymph juvenile-hormone-binding protein from Galleria mellonella (L)

A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile-hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20-25-kDa peptides. The rate of proteolysis of juvenile-hormone-binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone-binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission.     

Classification of Seven Species of Cactaceae Based on Their Chemical and Biochemical Properties

Fast protein liquid and ion-exchange chromatography, fluorescence, FT-IR spectra, and elemental and electrophoretic analyses were used to characterize proteins from seven species of Cactaceae, which can be divided into three groups based on their chemical and biochemical properties. Some cactus juice proteins precipitated by ammonium sulfate yielded complex electrophoretic patterns where the major bands correspond to approximately 24,000 Da and 32,000 Da in the presence of sodium dodecyl sulfate (SDS). The SDS-polyacrylamide gel electrophoretic (PAGE) patterns did not differ by the year of sample collection (1986 and 1992). Chromatographic analysis showed that protein characterization of cactus juices may be useful in cactus taxonomy at the family level. Fluorescence emission and FT-IR spectra of studied cacti species were measured to compare protein structure. Differences in the emission peak response and fluorescence intensity, as well as the changes in amide band content were found.     

Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots

Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.     

Fractionation and electrophoretic patterns of seed protein of Opuntia genus. A preliminary survey as a tool for accession differentiation and taxonomy

At present, little is known about Opuntia seed proteins and their contribution to the characterization and taxonomy of genotypes belong to this genus. The variation among 102 accessions of Mexican Opuntia was studied using electrophoretic patterns by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of their seed total proteins (STPs) and seed storage proteins (SSPs). Albumins and globulins were the most abundant protein fractions, with contents varied from 2.6 to 11.9 mg/mL and 2.6–9.5 mg/mL, respectively. These were followed by glutelins (2.3–8.5 mg/mL) and prolamins as the lowest (1.1–7.9 mg/mL) of the four protein factions. On the other hand, STPs content varied between 1.13 and 7.12 mg/mL. Regardless of variations in protein content estimated in seeds, the electrophoretic patterns of STPs and SSPs, as outcome of their SDS–PAGE, were not so variable. Furthermore, the individual analysis of each STPs or the SSPs analyses, separately, were not enough to discriminate all accessions, since it was necessary to combine the data resulting from all protein profiles together to differentiate all Opuntia genotypes. The UPGMA analysis indicated that there is no separation between accessions of species of the prickly pear (sweet fruits) and “xoconostle” (acidic fruits), even though the latter were grouped together. Based on biochemical markers analyzed herein, the need for revision of taxonomic assignment of genotypes belonging to the genus Opuntia is suggested.     

A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Two-dimensional electrophoretic analysis of spore proteins of the Microsporida

Microsporida are potentially useful as biological control agents for insects of economic and medical importance. Prior to their responsible use, however, an accurate and reliable means of identification to the species and subspecies level is required. Current methods used for identification are not adequate, due to variability of identifiable characters and to the occurrence of dimorphism. Recently, progress has been made in the use of biochemical characteristics to support the more traditional methods of distinguishing between morphologically similar species. We report on an improved method of characterization of microsporidan spore proteins, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). This method increased the number of spore polypeptides resolved from Nosema locustae spore protein extracts 2-3-fold over 1-dimensional PAGE. Also, each of the 2D-PAGE spore protein fingerprints of the species examined, namely Nosema locustae, Nosema bombycis, and Vairimorpha necatrix, were unique and differences in their spore protein composition were easily determined. The major structural proteins of Nosema locustae spores co-electrophoresed with alpha and beta tubulin from calf brain and had similar pI and molecular weight values as reported for tubulin in other species. Each species' 2D-PAGE fingerprint contained a few polypeptides that were present in relatively high concentration and these polypeptides may represent the major proteins of the structural components of the spore.     

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.     

Bean arcelin

Summary Crude proteins from seeds of wild bean accessions of Mexican origin were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE). Several accessions had electrophoretic patterns showing unique protein bands. When analyzed by two-dimensional isoelectric focusing (IEF)-SDS/PAGE, four protein variants which had electrophoretic mobilities similar to each other but different from the other major seed proteins, phaseolin and lectin, were observed. All four variants, which have not been described in cultivated beans, were tentatively named arcelin proteins and designated as arcelin 1, 2, 3 and 4. Arcelins 3 and 4 had polypeptides that comigrated on two-dimensional gels and these variants occurred in accessions that were collected in the same location. Analysis of single F₂ seeds from crosses among arcelin-containing lines and from crosses between cultivated beans lines without arcelin and arcelin-containing lines revealed that differences in arcelin polypeptide expression were inherited monogenically. The alleles for different arcelin variants were codominant to each other and dominant to the absence of arcelin. The gene(s) controlling arcelin proteins were unlinked to those controlling phaseolin expression and tightly linked to genes controlling the presence of lectin proteins (< 0.30% recombination). The possible origins of arcelin genes and their potential role in bruchid resistance are discussed.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate