Native Protein Electrophoresis Research Articles

Methods for studying protein targeting to and within the chloroplast.

Distinct protein complements impart each of the chloroplast's three membranes and three aqueous spaces with specific functions essential for plant growth and development. Chloroplasts capture light energy, synthesize macromolecular building blocks and specialized metabolites, and communicate environmental signals to the nucleus. Establishing and maintaining these processes requires approximately 3000 proteins derived from nuclear genes, constituting approximately 95% of the chloroplast proteome. These proteins are imported into chloroplasts from the cytosol, sorted to the correct subcompartment, and assembled into functioning complexes. In vitro import assays can reconstitute these processes in isolated chloroplasts. We describe methods for monitoring in vitro protein import using Pisum sativum chloroplasts and for protease protection, fractionation, and native protein electrophoresis that are commonly combined with the import assay. These techniques facilitate investigation of the import and sorting processes, of where a protein resides, and of how that protein functions.

Structural insights into Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase: oligomeric assembly.

The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 2.7.7.1). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plasmodium falciparum (PfNMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the PfNMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-PfNMNAT recombinant protein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution of an active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-PfNMNAT protein.

Application of native agarose gel electrophoresis of serum proteins in veterinary diagnostics

Abstract Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.

31 - Cytochrome P450 is the Source of NADPH-Dependent Signal in Cell-Free Assays for Nox Activity

Measuring Nox-derived reactive oxygen species is a constant challenge in redox biology. All probes available display limitations regarding sensitivity, specificity or demand highly specialized technique. To overcome such limitations, numerous studies have used NADPH-stimulated assays in membrane fractions to address Nox activity. In our previous work we found an unchanged activity in a triple Nox knockout (Nox1-Nox2-Nox4-/-) compared to its wild type. Moreover, results in intact cells overexpressing Noxes could not be recapitulated in NADPH-stimulated membrane assays showing that such assays are unable to measure Nox activity. Here we further investigate and validate the real source of NADPH-depend signal in membranes. By combining native protein electrophoresis, NADPH-stimulated assays and mass spectrometry we found mitochondrial proteins and cytochrome P450 as possible candidates responsible for the signal. Cells lacking functional mitochondrial complexes (I, III, IV and IV) showed no difference in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases and dehydrogenases are unlikely to generate the signal. However, in microsomes overexpressing P450 proteins there was a remarkable signal upon NADPH stimulation in lucigenin assay but also with LO12 and DHE. Knockdown of P450 reductase by CRISP/Cas9 technology abolished the signal in NADPH-stimulated assays showing that cytochrome P450 system accounts for the majority of the signal which has been for so long addressed to NADPH oxidases.

A novel method to predict protein aggregations using two-dimensional native protein microfluidic chip electrophoresis

A microfluidic chip native protein electrophoresis was established to predict protein aggregation.

High resolution two‐dimensional electrophoresis of native proteins

Blue native PAGE (BN-PAGE) is a powerful method to separate protein complexes while preserving their native state. However, the resolution of the method is limited as complexes with similar molecular masses cannot be resolved. Here we describe native 2DE using immobilized pH-gradients in combination with BN-PAGE to resolve protein complexes by their pI and molecular mass. This method enables electrophoretic separation of proteins between pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spots at large molecular weight were identified using MS to confirm potential protein complexes. Several protein complexes could be identified, most prominent GroEL in complex with GroES, parts of the ribosomal machinery and membrane transport system. In summary, this method enables easy high-resolution electrophoretic separation of protein complexes.

STIM1 Enhances SR Ca2+ Refilling through Activating SERCA2a in Rat Ventricular Myocytes

In cardiac ventricular myocytes, the function of “stromal interaction molecule 1” (STIM1), an ER/SR Ca2+ sensor, is still a mystery regarding its localization, mobilization, and Ca2+ signaling regulation. Here, adult rat ventricular myocytes freshly isolated or in primary culture were examined. Endogenous STIM1 was found by immunofluorescence and shown to be distributed mainly within the Z-disk. Using mcherry tagged expression of STIM1, we found that re-distribution of STIM1 does not occur after SR Ca2+ depletion (using thapsigargin, 2 μM) nor does SR Ca2+ depletion affect STIM1 movement within the SR when evaluated with fluorescence recovery after photobleaching (FRAP). Consistent with this result, native protein electrophoresis showed that STIM1 exists mainly as an oligomer, which is not altered upon SR Ca2+ depletion. Additionally, no store-operated Ca2+ entry (SOCE) or Ca2+ release-activated Ca2+ current (ICRAC) was observed in control or STIM1 overexpressing ventricular myocytes. The overexpression of STIM1 did, however, have dramatic consequences in ventricular myocytes: The SR Ca2+ content did increase as did SR Ca2+ leak. Parallel investigations indicated that STIM1 physically binds to phospholamban (PLN), suggesting that overexpressed STIM1 may activate SERCA2a by regulating PLN and this may underlie the increase in SR Ca2+ content. The molecular signaling and regulatory pathways that may be involved in these pathways are discussed.

Serum biochemistry and native protein electrophoresis in diarrheic calves with arthritis

In this study, serum biochemistry and native protein electrophoresis in newborn calves with diarrhea and arthritis, were performed in order to evaluate the changes along with clinical findings for their possible application in the diagnosis and prognosis of disease. Based on clinical examination, animals were allotied into two groups comprising either diseased or healthy animals. Urea, creatinine, ALT, AST, LDH, albumin, total protein, glucose, total cholesterol, uric acid and iron levels were determined in the sera. Serum protein native polyacrilamide gel electrophoresis (nPAGE) was performed followed by protein band ratio estimation supported with densitometry at 596 nm. Differences between the average mean of healthy and diseased animals were compared statistically (Kruskal-Walley test). In this study a decrease in serum glucose and cholesterol values (p<0.001), increase in urea, LDH levels and α1-and α2-globulin levels (p<0.01 and p<0.05 respectively) were found to be associated with the disease. As a result, the observed significant changes in biochemical parameters and clinical investigation in calves, suggesting acute inflammation causing the decrease in glucose and increase in α-globulins, may be of prognostic value.

Blue native protein electrophoresis for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins

Morphogenesis of the mouse polyomavirus virion is a complex and not yet well understood process. Nuclear lysates of infected cells and cells transiently producing the major capsid protein (VP1) of the mouse polyomavirus and whole-cell lysates were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) to characterize the participation of cellular proteins in virion precursor complexes. Several VP1-specific complexes were found by immunostaining with the anti-VP1 antibody. Some of these complexes contained proteins from the heat shock protein 70 family. The BN-PAGE was found to be a useful tool for the identification of protein complexes by immunostaining of separated cell lysates. However, whole-cell lysates and lysates of isolated nuclei of cells infected with polyomavirus appeared to be too complex for BN-PAGE separation followed by mass spectrometry. No distinct bands specific for cells infected with polyomavirus were detected by Coomassie blue stained gels, hence this method is not suitable for the discovery of new cellular proteins participating in virion assembly. Nevertheless, BN-PAGE can be valuable for the analyses of different types of complexes formed by proteins after their enrichment or isolation by affinity chromatography.

Protein composition of tobacco leaves with antiviral resistance induced by activators of defense responses and TMV infection

In tobacco (Nicotiana tabacum L.) plants of hypersensitive cv. Samsun NN, a capability of necrosis lesion formation and protein patterns were studied after induction of antiviral resistance by defense responses activators (DRA) (arachidonic acid, ubiquinone 50, and vitamin E) and by infection with tobacco mosaic virus (TMV). DRA and TMV improved both local and systemic leaf resistance to TMV. Native protein electrophoresis demonstrated differences in the composition of leaf proteins extracted under acidic and alkaline conditions. SDS-PAGE revealed proteins accumulated during the development of systemic antiviral resistance after lower leaf treatments with DRA and of local resistance induced by pretreatment with TMV. It was shown that various DRA affected protein patterns similarly, whereas TMV infection resulted in other changes. It is supposed that different pathways function in tobacco plants during induction of systemic resistance by DRA and TMV infection.

Analysis of vicilin (7S)‐class globulin in cocoa cotyledons from various genetic origins

AbstractCocoa cotyledons contain vicilin (7S)‐class globulin (VCG), a major storage protein. It is the native source of oligopeptides and free amino acids which have been identified as precursors of cocoa‐specific aroma and are formed through proteolysis during fermentation. High‐resolution electrophoresis of native proteins isolated from ripe, unfermented cocoa cotyledons harvested from different cultivars was used to determine genetic differences of the genotypes. Flavour differences have been reported to exist after standard fermentation in cocoa beans harvested from various genotypes. In this paper, SDS‐PAGE and 2D IEF/SDS‐PAGE polypeptide separation patterns are shown which were separately isolated from cotyledons of various genetic origins. Cotyledons from three cultivars belonging to genetically distant varieties and a hybrid, Criollo, Forastero and Trinitario, did not reveal any analytical identity differences of VCG subunit polypeptide bands. Additionally, proteins of cotyledons harvested from three of those clones which were reported to produce genotype‐specific flavour differences in raw cocoa after standard fermentation, SCA 12, UIT1 and PBC 140, when analysed in the same way, did not indicate differences. Thus the cotyledon storage proteins from various genetically different cocoa trees are, within methodological limits, the same. We conclude that aroma differences in raw cocoa harvested from various genotypes are the result of other genetic, physiological or curing‐related factors, but are not due to genetic differences of aroma precursors derived from storage proteins.© 2002 Society of Chemical Industry

The Association of Plant Stress Uncoupling Protein CSP 310 with Winter Wheat Mitochondria in vitro during Exposure to Low Temperature

Summary Plant stress uncoupling protein CSP 310 has been found to be associated with isolated winter wheat mitochondria during incubation in vitro at 0°C for 30 min. Pronase E treatment shows that this protein is localised outside of the mitochondria. It was found that CSP 310 is associated with mitochondria inactivated by CCCP and KCN. Electrophoresis of native proteins shows that if the native form of CSP 310 is associated with intact mitochondria then CSP 310, after its dissociation to 66 and 56kDa subunits, is associated with mitochondria inactivated by CCCP and KCN.

Isozyme pattern of glycogen phosphorylase in the rat nervous system and rat astroglia-rich primary cultures: electrophoretic and polymerase chain reaction studies.

Of the three isozymes of glycogen phosphorylase (GP) known, the brain (B) and muscle (M) isoforms have been reported to occur in brain. We investigated the regional and cellular occurrence of the three isozymes in various parts of the rat nervous system, fetal brain and astroglia-rich primary cultures by means of electrophoresis of native proteins with subsequent activity stain and by reverse transcriptase polymerase chain reaction. In the cortex, cerebellum, olfactory bulb, brainstem, spinal cord and dorsal root ganglia, both mRNA and enzyme protein were found for the B and M isozymes. In addition, the liver (L) isoform mRNA was detected in fetal brain and cultured astrocytes. Our studies indicate that there is no regional difference in distribution pattern between brain regions, spinal cord and dorsal root ganglia. In immature brain and cultured glial cells, the additional presence of the L isozyme is possible. These results support the idea that astrocytes express two or even three GP isozymes simultaneously.

Involvement of multiple copper/topa quinone-containing and flavin-containing amine oxidases and NAD(P) + aldehyde dehydrogenases in amine degradation by filamentous fungi

Eighty-five different fungal strains were screened for the presence of amine oxidase after it was induced by n-butylamine, methylamine or spermine. Amine oxidase activity was detected in all Aspergillus, Penicillium, and most of Monascus strains, as well as in Pullularia, Doratomyces, Armillaria, Fusarium, Gibberella, Sporotricus, Trichophyton, Beauveria, Caldariomyces, Helicostylum, and Trichoderma, but not in Mucor, Neurospora, Syncephalis, Keratinomyces, Eurotium, Zygorhynchus, or Cladosporium. By electrophoresis of native proteins with amine oxidase activity staining, two to three different enzymes were found in most of the strains. Predominant among these were copper-containing amine oxidases (EC 1.4.3.6), which were detected by Western blotting with polyclonal antibody against copper amine oxidase from Aspergillus niger AKU 3302. On SDS-PAGE, most of the enzymes showed bands corresponding to a molecular mass of about 75 kDa. Enzymes that did not react with the antibody against copper amine oxidase did not react either with the antibody against flavin-containing monoamine oxidase from Micrococcus luteus. However, the enzymatic activities of extracts that contained non-copper amine oxidases was substantially decreased by pargyline, an inhibitor specific to flavin-containing amine oxidases. Therefore, these enzymes are thought to belong to the class of flavin-containing amine oxidases (EC 1.4.3.4.). Besides amine oxidase activity, increased aldehyde dehydrogenase activity was found in selected strains after induction with amines, from which it is supposed that amines are degraded via aldehydes to corresponding carboxylic acids. Two alternate metabolic pathways are suggested for methylamine and for larger aromatic amines.

Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.

A rapid purification procedure produces milligram amounts of the T7 gene 4A' primase/helicase, 4B helicase, and the wild-type 4AB proteins expressed from the clones described in the accompanying paper (Rosenberg, A. H., Patel, S. S., Johnson, K. A., and Studier, F. W. (1992) J. Biol. Chem. 267, 15005-15012). Purified 4A' protein (in which the wild-type methionine at amino acid 64 has been replaced by leucine to eliminate the 4B initiation codon) appears to be equivalent to the wild-type 4A protein in primase, helicase, and NTPase activities. Gel filtration chromatography and polyacrylamide gel electrophoresis of native proteins indicate that the 4A' and 4B proteins form homodimers and heterodimers in solution. Heterodimer formation presumably accounts for an observed 3-fold increase in the primase activity of 4A' upon addition of 4B that lacks primase activity of its own. Steady-state k(cat) and Km values for hydrolysis of the nucleoside triphosphates ATP, dATP, dTTP, and dGTP were measured for 4A', 4B, 4A'B (1:1), and wild-type 4AB (1:2) proteins. The dependence of the dNTPase activities on the concentration was hyperbolic, suggesting single or noncooperative binding sites, whereas ATPase activity was sigmoidal, suggesting more than one ATP binding site. The k(cat)/Km ratios for hydrolysis of the dNTPs by the four protein preparations were within a factor of 6 of each other. The 1:1 mixture of 4A'B had the highest k(cat)/Km ratios, with a preference for dATP and dTTP.

Identification of an ovalbumin-like protein in egg-white of turtle and alligator

1. 1. Egg-white proteins from Pseudemys scripta and Alligator mississippiensis do not exhibit a rapid migrating protein in high concentration equivalent to bird ovalbumin when electrophoresed in starch and non-denaturing polyacrylamide. 2. 2. Egg-white proteins of both reptiles had a lightly staining band of 45,000 molecular mass. 3. 3. When blotted to nitrocellulose membrane after denaturing (sodium dodecyl sulfate) polyacrylamide electrophoresis, and treated with polyclonal anti-bodies directed toward ovalbumin, an immunostaining reaction occurred with this protein from P. scripta and alligator. 4. 4. An ovalbumin-like protein, based on molecular mass and immunological similarities, occurs in eggs of these reptiles although its presence was not detectable by native protein electrophoresis.

Electrophoresis of Native Proteins on Agarose Gels Containing Volatile Buffers

Abstract Protein mixtures were resolved under non denaturing conditions on agarose gels containing the volatile buffer ammonium bicarbonate. Proteins were detected on a guide strip of the gel by a rapid staining method and the corresponding bands cut from the gel and eluted by centrifugation. The proteins were then obtained by lyophilization. Native proteins in the gel could be rapidly blotted onto nitrocellulose paper by capillary transfer.

セ・ア膜による２次元電気泳動法

セ・ア膜による２次元電気泳動法

Purification of Avidin by Cation Exchange, Gel Filtration, Metal Chelate Interaction and Hydrophobic Interaction Chromatography

Avidin is commonly recovered in crude form from egg albumen by treatment with a cation exchange resin or by solvent or salt precipitation. Many methods are available for subsequent avidin purification. In this study, carboxymethyl cellulose (CMC) cation exchange, gel filtration, metal chelate interaction, aliphatic hydrophobic interaction and aromatic hydrophobic (Phenyl Sepharose) interaction chromatography each resulted in a substantial increase in avidin purity. In terms of resin capacity, yields and avidin purity, however, CMC cation exchange chromatography was superior. A comparison of sodium dodecyl sulfate polyacrylamide gel electrophoresis and native protein electrophoresis profiles gave evidence of protein-protein interaction between avidin and lysozyme in partially-purified avidin preparations. This interaction may also occur between the native proteins in the egg white, but this has not been demonstrated with certainty.

Two-dimensional gel electrophoresis method for investigation of human plasma proteins: detection of subtle changes during filtration leukapheresis.

Special problems are associated with analysis of human plasma proteins by standard "high-resolution" two-dimensional gel electrophoresis methods in which isoelectric focusing is followed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels (SDS-PAGE). Individual plasma proteins are often separated into overlapping groups of multiple spots, and identification of individual spots is further confounded by genetic variation. Analytical recovery of components of high molecular mass is also low or variable. These problems may be reduced or overcome by use of a "low-resolution" method consisting of electrophoresis of native proteins at pH 8.6 in an agarose gel followed by SDS-PAGE without added reducing agent. About 60 proteins can be resolved, most as single spots. About 25 of these proteins have been "mapped," and tentatively identified. We have examined 119 plasma samples taken from six donors who were undergoing filtration leukapheresis and 10 donors who were undergoing centrifugation leukapheresis or plateletpheresis. In all cases, passage of blood through a nylon filter induced a significant increase in a doublet of spots tentatively identified as complement component C3c. This was detected in the effluent from the filter throughout the first 30 min of filtration, and to a lesser extent in the venous blood. These spots were not induced by the centrifugation procedures. One filtration donor also showed increased acute-phase proteins 24 h after the procedure.