Two-dimensional electrophoretic analysis of encephalomyocarditis viral proteins

All four capsid proteins of encephalomyocarditis virus and the precursor to two of these were resolved from purified virions with two-dimensional gel electrophoresis. In addition, all of the known stable virus-specific proteins found in infected cells, but not the primary and intermediate precursor proteins, could be resolved with these techniques.     

Two-dimensional electrophoretic analysis of the regulation of SOS proteins in three ssb mutants

A previously undescribed mutation in the ssb gene, which codes for a major single strand DNA binding protein essential for DNA repelication, was mapped on the Escherichia coli Chromosome. Three ssb mutants were analyzed under parallel physiological conditions for the induction of SOS proteins (products of recA, uvrA, and an unknown gene), the production of mutants, the induction of lambda prophage, and sensitivity to DNA damaging agents. Two-dimensional electrophoretic techniques were used to quantitate changes in the rate of synthesis of proteins. The previously unpublished position of the uvrA gene-product in the two-dimensional matrix of E. coli proteins was described. These ssb strains exhibited varying sensitivities to ultraviolet irradiation and methylmethane sulfonate that correlated with the rate of constitutive synthesis of SOS proteins, spontaneous commitment to virulent growth of lambda lysogens, and elevation of endogenous mutation rates.Dedicated to the memory of Roger Y. Stanier: to his fascination for diverse microbial lifeforms that catalyzed curiosity in his associates, to his intellectual aura that elicited deep respect, to his pursuit of scientific truth that promoted the highest research ethics, to his friendly nature that encouraged my growth as a scientist and enkindled my love for Roger     

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin

In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.     

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Two-dimensional protein electrophoretic analysis of postponed aging inDrosophila

Five populations ofDrosophila melanogaster that had been selected for postponed aging were compared with five control populations using two-dimensional protein gel electrophoresis. The goals of the study were to identify specific proteins associated with postponed aging and to survey the population genetics of the response to selection. A total of 321 proteins were resolvable per population; these proteins were scored according to their intensity. The resulting data were analyzed using resampling, combinatoric, and maximum parsimony methods. The analysis indicated that the populations with postponed aging were different from their controls with respect to specific proteins and with respect to the variation between populations. The populations selected for postponed aging were more heterogeneous between populations than were the control populations. Maximum parsimony trees separate the selected populations, as a group, from their controls, thereby exhibiting a homoplastic pattern.     

Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.     

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.     

Two-dimensional spectroscopy of electrophoretic gels

Optical techniques are described which permit one to analyze two-dimensional electrophoretic gels in a fashion which is analogous to the one-dimensional spectroscopy of solutions. In the methods described, an electrophoretic gel is irradiated with monochromatic light and isozyme patterns are detected by the absorption of light or the fluorescent emission of light. The system described can both generate and detect monochromatic light in a range from 200 to 1100 nm. Without the use of histochemical stains, several isozymes have been visualized by purely optical means. Five methods for the visualization of lactate dehydrogenase and five methods for the demonstration of trypsin isozymes are described. In addition, general methods have been formulated for hydrolases and oxidases. Gel spectroscopy should permit the investigation of a wide range of new isozymes.This work was supported in part by NIH Grants CA 19017 and GM 21433.     

Two-dimensional electrophoretic analysis of major phosphoproteins of the sea urchin, Arbacia punctulata

Phosphoproteins of hatched blastulae, gastrulae, and pluteus larvae of the sea urchin, Arbacia punctulata, were labeled in vivo with [32P]O4 and analysed by 2-dimensional polyacrylamide gel electrophoresis and autoradiography. At least 60 phosphoproteins were resolved. Some of these showed different relative intensities of labeling at the embryonic periods monitored. Some embryonic phosphoproteins were characterized by cell fractionation and by comparing autoradiograms with Coomassie-blue staining patterns and [35S]methionine labeling patterns. Neither actin nor tubulin phosphorylation was detected. No differences in phosphorylation were detected in dissociated and partially reassociated blastula cells relative to each other and to intact embryonic controls.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

A simplified method for the electrophoretic analysis of hair proteins

Hair proteins have been analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using a simplified method without S-carboxymethylation, protein fractionation and lyophilization. The molecular weights of the proteins have been determined and human, rat, guinea pig, rabbit, gerbil, cow and sheep hair compared. These molecular weight values are consistent with those obtained using physical methods as compared to anomalously high values previously reported following electrophoresis of S-carboxymethylated hair proteins. Additional high molecular weight proteins, possibly of a dimeric nature, have also been detected.     

Two-dimensional gel electrophoretic analysis of the HindIII 1.8-kb repetitive-sequence family in the human genome

The structure and organization of a human repetitive DNA family containing the HindIII 1.8-kb repetitive sequence were studied, using two-dimensional (2D) gel electrophoresis. The HindIII 1.8-kb sequence proved to be part of a repetitive sequence about 5 kb long and interspersed on the genome. The long repetitive sequence family contained several subgroups, as based on polymorphism of the restriction site. Recombinant phages containing the long repetitive sequence were isolated from the human genomic DNA library. Heteroduplex and restriction analysis showed that the structure of the repetitive sequence carried by the phages was close to that expected from 2D gel electrophoretic analysis. The 2D gel electrophoretic analysis was shown to be a reliable and useful approach for surveying and mass analysis of repetitive sequence families.     

An electrophoretic analysis of the basic nuclear proteins of ram spermatids

By a two-step separation procedure, round and elongated spermatid populations were obtained with purities of 90–97% and an intermediate spermatid population with a purity of 85%. Cells of ram testes were first separated at unit gravity and 5 spermatid fractions were collected. They were centrifuged for 23 min at 4 000 g in linear and non-linear isotonic polymer-free colloidal silica gradients. Improvements were made in centrifuge tubes and in gradient collection for optimum results. Fractions were analysed using a phase contrast microscope for cell counting and determination of cell viability. True cell density was measured and found to increase during late spermiogenesis from 1.045 g/cm³ for round spermatids to 1.152 g/cm³ for testicular spermatozoa, thus reflecting an increase in the nuclear density.     

Two-dimensional electrophoretic analysis reveals that lipid rafts are intact at physiological temperature

Different proteins are found in lipid rafts depending on the isolation method. For example, insulin receptor was predominantly found in lipid raft fractions prepared from HepG2 cells with Brij 35, but were not present in lipid rafts isolated with Triton X-100. In order to assess the effect of detergent type and temperature on raft isolation, raft proteins from HepG2 cells were analyzed by two-dimensional (2-D) electrophoresis. More raft protein spots appeared when rafts were isolated by Brij 35 than by Triton X-100. In addition, more raft proteins were found when isolated at 37 degrees C than at 4 degrees C, indicating that lipid rafts are much more stable at physiological temperature (37 degrees C) in the presence of detergents. Indeed, lipid-modified proteins, such as Src and Lyn, were found in raft fractions even when detergent-resistant rafts were isolated at room or physiological temperature. The 2-D gel profile of raft proteins isolated with detergent-free (high-pH/carbonate) method was considerably similar to that of detergent-resistant raft proteins but contained a greater number of distinct protein spots. Whereas many detergent-resistant raft proteins disappeared upon cellular exposure to methyl-beta-cyclodextrin, high pH/carbonate-resistant raft proteins did not, suggesting that many of proteins isolated by high pH/carbonate could be contaminants. Considering these data, we conclude that liquid-ordered state of detergent-resistant lipid rafts is not destroyed at physiological temperature.