Detergent Gel Electrophoresis Research Articles

Knockout of an Azorhizobial dTDP-L-Rhamnose Synthase Affects Lipopolysaccharide and Extracellular Polysaccharide Production and Disables Symbiosis with Sesbania rostrata

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium ion-dependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose- and Ca2(+)-dependent manner and stimulated NAD kinase from peas in a dose-dependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzyme-linked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pI of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.

Use of mild detergent gel electrophoresis for isolation and characterization of the kidney brush border d-glucose transporter

Gradient gel electrophoresis was performed under mild detergent conditions to separate pig kidney brush border membrane proteins and to identify the smallest functional molecular protein entity of the d-glucose transporter. The various protein bands obtained from the nondenaturing gel system in a semipreparative scale were eluted by electrodialysis. These proteins were then reintegrated into proteoliposomes and tested for d-glucose-inhibitable [ 3H]phlorizin binding. The d-glucose transporter had a molecular mass of 70 kDa in mild detergent electrophoresis conditions and in subsequent SDS analysis.

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis.

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions.

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.

Interactions of heme proteins with hydrogen peroxide: Protein crosslinking and covalent binding of benzo[ a]pyrene and 17β-estradiol

This work reveals two biochemical effects of hydrogen peroxide treatment on hemoglobin, myoglobin, and cytochrome c. First, these heme proteins rapidly formed covalently crosslinked dimers and polymers detectable by detergent gel electrophoresis. Second, when treated in the presence of radioactive benzo[ a]pyrene or 17β-estradiol, the heme proteins became covalently labeled. Nonheme proteins exhibited both cross-linking and radioactive labeling upon peroxide treatment in the presence but not the absence of heme protein or free hemin. Benzoyl peroxide or glucose and glucose oxidase effectively replaced direct addition of hydrogen peroxide. These results indicate that adventitious peroxidase activity expressed by oxygen carrying and electron transport proteins yields active oxygen species that can damage these heme proteins and nearby macromolecules, a possible biochemical mechanism for the lethal and other deleterious intracellular effects of perioxide.

Histone subtypes and switches in synthesis of histone subtypes during Ilyanassa development

The histones of the mud snail ( Ilyanassa obsoleta) have been examined at various stages during development, from early embryos to differentiated larvae. Histones were visualized by SDS electrophoresis and by nonionic detergent gel electrophoresis. For several developmental stages the synthesis of putative histone subtypes was monitored by fluorography. During cleavage little histone synthesis is detectable indicating that the histones of new chromatin of early Ilyanassa embryos come from a pool synthesized during oogenesis. We have found several H1 forms which we have tentatively designated as subtypes. Our results indicate that there is a stage-specific pattern of synthesis of these proteins during development. A prominent histone component of veliger larvae which migrates in the H2A-H2B region of an SDS-gel shows no or very little synthesis during embryogenesis. This component may contain both an H2A and an H2B subtype. In addition, we have resolved utilizing nonionic detergent gels one further form each of H2A and H2B which are synthesized during embryogenesis. Finally, we have found two Ilyanassa histones which have mobilities and staining properties similar to late sea urchin H2B subtypes. These proteins are synthesized during early embryogenesis. By the larval stage they are no longer synthesized at detectable levels.

Planorbis corneus haemoglobin : Circular dichroism and susceptibility to proteases

The purified haemoglobin of Planorbis corneus was subjected to protease digestion and the resulting products characterised by gel filtration and detergent-gel electrophoresis. Small functional subunits of molecular weights approximately 20 000 were obtained corresponding to a single haem group, but multiples of this unit were also always obtained even at high proteolytic enzyme : haemoglobin ratios. This suggested that the subunits of the native molecule (one-tenth containing perhaps ten O 2-binding sites) were made up of single-binding site domains linked by regions of polypeptide chains having different susceptibilities to proteases. The far ultraviolet CD of the native haemoglobin indicated the presence of a high helix content (75–80%) in the protein. The near ultraviolet and visible CD spectra of oxy- deoxy-, and CO-haemoglobin were reported. Planorbis haemoglobin CD was more like that of vertebrate haemoglobins than that of annelid haemoglobins. Nevertheless the Soret CD of Planorbis oxyhaemoglobin had only about half the rotational strength of that of human haemoglobin A, and was halved again upon removal of the ligand. Also in contrast to Lumbricus and human haemoglobins there was only a small decrease in rotational strength in the 260 nm band when Planorbis oxyhaemoglobin was deoxygenated.

Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus.

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.

Purification of the Ca2+-stimulated ATPase activator from human erythrocytes. Its membership in the class of Ca2+-binding modulator proteins

The activator of the Ca2+-stimulated ATPase of erythrocyte membranes was purified 13,000-fold to homogeneity from human erythrocytes. The protein gave a single band upon electrophoresis both with and without detergent, and upon isoelectric focusing. This protein was compared with Ca2+-binding modulator proteins from bovine brain and rat testis. All three proteins were homogeneous and co-migrated on electrophoresis both in the presence of detergent and without detergent at pH values on both sides of the isoelectric point of the protein. The amino acid compositions of the three proteins were nearly indistinguishable, and all three proteins contained 1 residue of the unusual amino acid, trimethyllysine. All three were also indistinguishable as measured by their ability to further stimulate the Ca2+-stimulated ATPase of human erythrocyte membranes. Thus, we conclude that they represent functionally the same protein. Upon storage of all three proteins, a second band was detectable by detergent gel electrophoresis; the biochemical activity and the behavior on nondetergent gels were not changed. The presence of this second band is probably responsible for previous reports of differences between the rat testis and bovine brain modulator protein. The possibility is discussed that this protein is a general intracellular Ca2+ receptor, which mediates the activities of Ca2+ as an intracellular messenger.

The spontaneous release of a high-molecular-weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells.

The spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface-bound humoral immune components are apparently released in the form of a high-molecular-weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes. IgG is localized on the tumor cell surface in a highly heterogenous pattern with the appearance of patches and caps in some cells as shown by immunofluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high-molecular-weight aggregates of cell surface antigens into extracellular fluids is discussed.

Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.

Identification and subcellular distribution of adipocyte peptides and phosphopeptides.

Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.

Characterization of the extracellular haemoglobin of Haemopsis sanguisuga (L.)

The haemoglobin from the blood of the horseleech, Haemopsis sanguisuga (L.), had a sedimentation coefficient, SO20, w, of 59.11 +/- 0.55 S, and a molecular weight as determined by sedimentation equilibrium of 3.71 X 10(6)+/-9904 X 10(6). In the electron microscope the molecule appeared to be made up of two hexagonal plates, as is found with other worm haemoglobins, with dimensions 24.4+/-2.0 nm (across the hexagon) and 15.2+/-1.4 nm (height). The amino acid composition and spectrum were closely similar to those of the haemoglobins of other annelids (e.g. Lumbricus). The alpha-helical content, calculated from circular-dichroism measurements in the far-u.v. region, was 56-63%. The haem content was 2.49%, corresponding to a minimum molecular weight per haem group of 24 800, but detergent-gel electrophoresis indicated the presence of polypeptide chains of mol.wts. 12 600, 14 800, 15 500 and 25 100. The pH-induced dissociation of the native molecule yielded compotosol of Soya-bean root nodules.

Isolation and general characterization of myoglobin from the dolphin fish Coryphaena hippurus (L.)

1. 1. Myoglobin was isolated from the red skeletal muscle of the dolphin fish Coryphaena hippurus (L.). The myoglobin was freed from minor components by chromatography on DEAE-Sephadex. 2. 2. The myoglobin migrated as a single component in polyacrylamide gel electrophoresis. Sedimentation equilibrium indicated a homogeneous preparation. The molecular weight was determined by sedimentation equilibrium and by detergent-gel electrophoresis. 3. 3. Absorption maxima and molar absorption coefficients were determined for the ferrimyoglobin. The pK′ for the acid dissociation of the haem-bound water molecule in the ferryimyoglobin was shown to be 8·89. 4. 4. The amino acid composition was determined. The myoglobin showed a low histidine and a high aspartic acid content.

Isolation and characterization of two-copper subunits from Murex trunculus haemocyanin

1. 1. Functional subunits of about 50,000 molecular weight, containing two copper atoms, were obtained by limited subtilisin digestion of Murex trunculus haemocyanin. 2. 2. The subunits showed a single peak in gel filtration and sedimentation velocity experiments, but were heterogenous in polyacrylamide gel electrophoresis. 3. 3. The binding of oxygen by the subunits was non-co-operative, without a Bohr effect and with decreased affinity with respect to whole haemocyanin. 4. 4. Detergent-gel electrophoresis indicated for the subunits a quarternary structure of four polypeptide chains without interchain disulphide bonds. The polypeptide chains showed a very strong tendency to aggregate. Species of about 25,000 and 12,500 molecular weight were demonstrated by gel filtration after reduction and carboxymethylation of holo subunits.

Murex trunculus haemocyanin. I. Physical properties and pH-induced dissociation.

The native or whole molecule of the haemocyanin from the whelk, Murex trunculus had a sedimentation coefficient of 102.7 S, and combination of this with the diffusion coefficient of 1.03 × 10−7 cm2 s−1 led to a molecular weight of 8.85 × 106. The parameter β of Scheraga and Mandelkern was 2.14 × 106, and the frictional ratio f/f0 was 1.5 indicating that the molecule did not deviate markedly from the spherical. Electron microscopy showed that the molecule was a short cylinder, about 35 nm in diameter.When the pH was raised dissociation occurred into halves and then into smaller subunits which were referred to as “twentieths”, but which were heterogeneous. This dissociation took place at pH values greater than 7.6 in the absence of Mg2+, or at pH values near 10.0 in presence of 0.01 M MgCl2. Whole molecules were re‐formed from halves upon lowering the pH, but “twentieths”, once formed, did not re‐associate. The products of dissociation were separable by preparative ultracentrifugation and by zone electrophoresis. The proportion of wholes, halves, and twentieths formed was determined by the pH and Mg2+ concentration and was not influenced by rotor speed or by protein concentration. It was considered that there was not a dynamic equilibrium between wholes, halves and twentieths.In 6 M guanidine‐HCl in the presence of 2‐mercaptoethanol a single species of molecular weight about 220000 was formed. In detergent‐gel electrophoresis there was rather poor evidence for a component of molecular weight about 50000, which whould correspond with one oxygen‐binding site containing 2 copper atoms.

Characterization of the plasma membrane of Mycoplasma laidlawii: V. Effects of selective removal of protein and lipid

Purified plasma membranes of Mycoplasma laidlawii were subjected to proteolytic enzyme digestion and aqueous acetone extraction. Up to 80% membrane protein could be removed by pronase, with a concomitant decrease in density of residual banding material. This membrane residue was still organized in vesicles and showed a decreased thickness and contrast in electron micrographs. Despite degradation of all characteristic membrane proteins (as shown by detergent—polyacrylamide gel electrophoresis) some protein fragments were retained in vesicular residues. The amino acid composition of this material was indistinguishable from that of total membrane protein, arguing against any enrichment in hydrophobic protein regions. Membrane hexosamine was not solubilized by pronase or aqueous acetone extraction, and could be substantially purified by combining these techniques. Extraction with 90% aqueous acetone removed over 95% of labeled membrane lipid but left sedimentable, protein-rich membranous material with unit membrane morphology. Such lipid-depleted membranes banded in sucrose gradients at a density of 1.25 g/cm 3 as a single narrow band, in contrast to the isopycnic density of 1.17–1.18 g/cm 3 for native membranes. At low pronase concentrations, electrophoresis experiments suggested preferential pronase attack on some higher molecular weight membrane proteins; these same electrophoretic components were preferentially released at very low concentrations of sodium dodecyl sulfate,, indicating differences in the organization of proteins within the membrane. Analysis of membranes labeled with [ 14C]glucosamine or [ 14C]oleate by detergent-gel electrophoresis followed by gel fractionation and counting showed that all oleate-containing lipid moved as a single broad band, while glucosamine migrated as two bands, one extremely broad and heterogeneous.