Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Patterns Research Articles

Actinomyces slackii sp. nov. from Dental Plaque of Dairy Cattle

Five strains of actinomyces isolated from dental plaque of cattle formed a homogeneous group on the basis of Gram staining, colonial and cellular morphology, fermentation reactions, acid end products of metabolism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides, cell wall carbohydrates, and peptidoglycan and deoxyribonucleic acid mean base compositions. These organisms were closely related, as determined by deoxyribonucleic acid-deoxyribonucleic acid homology, which distinguishes them from strains of Actinomyces naeslundii, Actinomyces viscosus, Actinomyces denticolens, and Actinomyces howellii. A new taxon, Actinomyces slackii, is proposed for these isolates. The type strains of A. slackii is strain NCTC 11923.

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody.

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.

Subspecies-specific surface antigens of promastigotes of the Leishmania donovani complex

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of proteins and externally exposed labeled surface constituents were analyzed in promastigotes of three etiological agents of kala azar (Leishmania donovani, HS70 strain from India; L. chagasi, Imperatriz strain from Brazil; L. infantum, ITMPA K263 strain from Morocco and MO strain from France). Coomassie blue-stained gels showed similar protein patterns for L. donovani and L. chagasi and a more distinct one for L. infantum. Surface radioiodination with two different methods, lactoperoxidase and IODO-GEN, gave identical autoradiographic patterns for each parasite. Four major labeled proteins with apparent Mr values of 65,000, 60,000, 50,000, and 26,000 were detected in both L. chagasi and L. donovani. However, the radioiodinated polypeptide pattern of L. infantum only showed two major bands with an apparent Mr of 62,000 and a doublet of 26,000 to 23,000. Immunoprecipitation of detergent extracts of labeled promastigote subspecies with immune sera from rabbits immunized with either L. chagasi or L. infantum and from patients and mice infected with these two parasites, as well as with a monoclonal antibody against the surface of L. donovani promastigotes, demonstrated that the surface antigenic expression of L. infantum is different from that noticed in the two other subspecies, which are similar. Immunofluorescence experiments with some of these antibodies confirmed these results. The present findings should be considered in taxonomic and immunological studies in visceral leishmaniasis.

Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins.

Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60 micrograms) from the ear isolate also inhibited bactericidal activity in the respective immune serum. LPSs exhibited minimal inhibition (greater than 110 micrograms). Three human sera (two normal, one immune) were selectively depleted of 80% of antibody activity against OMPs (measured by enzyme-linked immunosorbent assay) by affinity chromatography using OMPs from the pulmonary isolate coupled to a solid phase. These OMP antibody-depleted sera also showed an 88% reduction of bactericidal activity against this strain. Immunopurified antibody against OMPs eluted from the solid phase was bactericidal.

Endosperm Protein Synthesis and l-[35S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[(35)S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[(35)S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing patterns for endosperm proteins were similar for field and cultured kernels throughout development. By 15 days, over 70% of the l-[(35)S]methionine taken up was present in endosperm proteins. Label incorporation visualized by fluorography generally followed the protein intensity of the stained gels. The high methionine content, low molecular weight zeins (i.e. 15 and 9 kilodaltons) were highly labeled. All of the radioactivity in hydrolyzed zein samples was recovered in the methionine peak indicating minimal conversion to l-[(35)S]cysteine. The procedure described here is suitable for long term culture and labeling experiments in which continued kernel development is required.

The reassociation of Lumbricus terrestris hemoglobin dissociated at alkaline pH.

The reassociation of the extracellular hemoglobin of Lumbricus terrestris (Mr approximately 3.9 X 10(6) ) at neutral pH, subsequent to its dissociation at pH above 8.0, was examined using gel filtration, ultracentrifugation, and scanning transmission electron microscopy. Gel filtration on Sephacryl S-200 at pH 6.8 of the hemoglobin exposed to pH above 8 showed the presence of four peaks: Ia, consisting of whole molecules, undissociated and reassociated, and smaller heme-containing fragments Ib (Mr approximately 3.0 X 10(5) ), II (Mr approximately 6.5 X 10(4) ), and III (Mr approximately 1.8 X 10(4) ). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that although the pattern of Ia was identical with that of the native hemoglobin, consisting of six polypeptide chains (I-VI), Ib appeared to have less of chains V and VI; II consisted of polypeptide chains II-VI, and III was identified as chain I. Lumbricus hemoglobin exposed to pH over the range 8.4 to 10.2 was subjected to gel filtration on Sepharose CL-6B and resolved into undissociated and dissociated fractions. The combined dissociated fractions, when brought back to pH 6.8 and subjected to gel filtration on Sepharose CL-6B and Sephacryl S-200, demonstrated that the extent of reassociation into whole molecules (IaR) varied from about 50% at pH 8 to 30% at pH 10.2. IaR possessed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern identical with that of the native hemoglobin. Ia, IaR, and Ib dissociated when exposed to alkaline pH; upon return to neutral pH, both Ia and IaR reassociated partially to whole molecules but Ib did not. These results suggest that reassociation comprises two pathways: one leading to the formation of IaR and a "dead-end" pathway, along which reassociation stops at the level of Ib. Digital image processing of scanning transmission electron micrographs of negatively stained native hemoglobin and IaR showed that the two molecules were very similar.

Inhibition of neutrophil function by fluid phase C3b of complement.

A high-molecular-weight fragment of C3 was isolated from normal human serum by column chromatography, was generated by incubation of serum at 37 degrees C with inulin, and was produced from highly purified C3 by limited digestion with trypsin. This product was shown to inhibit the antibacterial function of neutrophils by using Escherichia coli O75 as the main test organism. The inhibitor reacted with anti-C3b and anti-C3c, but not with anti-C3B (anti-native C3) or anti-C3a. The manner of preparation of the inhibitor, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, and the amino acid composition of the inhibitor indicated that it was fluid phase C3b. The inhibitor of neutrophil function (fluid phase C3b) was shown to bind to C3b receptors or acceptors on sheep erythrocytes in a model system.

Preparation and physicochemical and immunological characterization of polysaccharide-outer membrane protein complexes of Neisseria meningitidis

A crude complex containing group C polysaccharide, outer membrane proteins, and lipopolysaccharide (LPS) was isolated from the cell-free culture liquid of Neisseria meningitidis serogroup C, serotype 2a. Group C polysaccharide and LPS were removed from this complex, resulting in an outer membrane complex and a purified complex, respectively. Analysis by electron microscopy showed the outer membrane origin of the crude complex and the outer membrane complex, whereas such a structure was absent in the purified complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of the three complexes were identical. Pyrolysis-mass spectrometry data correlated well with those obtained by the biochemical assays and suggested a low LPS content in the purified complex and a low polysaccharide content in the outer membrane complex. The purified complex was shown to be nonpyrogenic and could be prepared with the same yield as that of purified polysaccharide. The immunogenic activities of the complexes were studied in mice. The antibodies were measured by the enzyme-linked immunosorbent assay; and the bactericidal antibody assay. All complexes induced immunoglobulin G antibodies to group C polysaccharide as well as to the serotype antigen, although the removal of polysaccharide and LPS resulted in a reduction of the immunogenic activities of outer membrane complex and purified complex, respectively. A second dose of all complexes produced a clear booster effect of both antibody responses. The antibodies were bactericidal.

Characterization of Lupine Proteins

ABSTRACTProteins of eight cultivars of two lupine species (Lupinus albus and L. angustifolius) were examined by several procedures. Differences in protein solubility between the species and among varieties within a species were observed. Defatting of the lupine flour altered protein solubility. Amino acid compositions of total protein and protein fractions showed only minor differences between the species and among varieties within a species (except for cv. Kali). Sodium dodecylsulfate polyacrylamide gel electrophoresis patterns of protein fractions revealed both qualitative and quantitative differences between species and among varieties.

Noncollagenous proteins in cartilage of normal subjects and patients with degenerative joint disease. A gel electrophoretic study.

Normal articular cartilage from subjects of various ages and cartilage from patients with degenerative joint disease were extracted with 4M guanidinium chloride. After dialysis against 8M urea pH 6.8, a 0.2M NaCl fraction was obtained by ion exchange chromatography on DE-52 in 8M urea. This fraction was concentrated, reduced, and analyzed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (7% gels). Six major noncollagenous protein bands (P1-P6) were found; 2 were identified as the link proteins. The approximate molecular weights of P1-P6 were: 87,000, 64,000, 56,000, 46,000, 41,000, and 27,000. A similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of P1-P6 was found in young baboons, in normal young and aged humans, and in patients with degenerative joint disease. Peaks corresponding to extracted collagen were decreased in older patients and increased in patients with degenerative joint disease, even those of advanced age.

Membrane composition and virus susceptibility of Acholeplasma laidlawii

The membrane composition of 11 strains of Acholeplasma laidlawii, including three strains persistently infected with mycoplasmaviruses MVL51, MVL2, and MVL3, was studied and correlated with mycoplasmavirus sensitivity. Membranes of the strains had similiar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, and all strains were inhibited by an antiserum produced against membranes from one of the strains. The amounts of integral membrane proteins solubilized by the nonionic detergent Tween 20 differed considerably. Therefore, characteristic crossed immunoelectrophoresis patterns were obtained for each strain. Strains persistently infected with MVL2 and MVL3 were notably different from the noninfected host. The ability to propagate any of the viruses was not correlated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis or crossed immunoelectrophoresis patterns. The persistently infected strains had a characteristic lipid composition. MVL51-resistant strains, including a resistant clone selected from a sensitive strain, were characterized by a large monoglucosyldiglyceride/diglucosyldiglyceride ratio and trace amounts of diphosphatidylglyceol (as opposed to the sensitive strains). Differences in lipid composition in A. laidlawii seem to affect the relationship between cells and viruses.

Detergent properties of water-soluble choline phosphatides. Selective solubilization of acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes.

Several analogs of lysolecithin were found to solubilize human erythrocyte ghosts comparably or even better than other detergents. Derivatives with aliphatic chains of 12 to 14 carbons were most effective. The phosphorylcholine detergents apparently possess low protein-denaturing properties, since they, for the first time, allowed the solubilization of enzymatically active acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes. The solubilized enzyme was not sedimented at 177,000 x g for 60 min and penetrated into Sepharose 6B gels. Low detergent concentration resulted in a selective extraction of the acyltransferase (about 70%) as compared to alkaline phosphatase, nucleotide pyrophosphatase, gamma-glutamyltransferase or Mg2+-ATPase (30 to 40%). The selectivity was reflected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of soluble and sedimentable membrane fractions; three bands of approximately 53, 84, and 94 x 10(3) daltons were enriched in the supernatants, whereas one band of about 68 x 10(3) daltons was concentrated in the pellet. The preferential extraction of acyltransferase may be related to particularly high affinity of lysolecithin analogs for this enzyme, which at higher concentrations was competitively inhibited by these detergents. The inhibitor constants ranged from 1400 micron for the C10 analog (ET-10-H) to 80 micron for the compound with 16 carbons (ET-16-H) per aliphatic chain.

Muscle surface membranes. Preparative methods affect apparent chemical properties and neurotoxin binding

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050–1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [ 3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using α-[ 125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p- nitrophenylphosphatase , higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfatepolyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.

Concanavalin A augments the turnover of electrophoretically defined thymocyte plasma membrane proteins

1. 1. Rabbit thymocytes were labelled in vitro with 14C-labeled amino acids either before or during stimulation with concanavalin A. 2. 2. Plasma membranes were isolated from control and stimulated thymocytes and their proteins fractionated by sodium dodecylsulfate-polyacylamide gel electrophoresis. 3. 3. No qualitative and quantitative differences between the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of membrane proteins from normal and concanavalin A-treated cells could be detected. 4. 4. Plasma membrane protein turnover is generally augmented by concanavalin A stimulation. 5. 5. The turnover of a sodium dodecylsulfate-polyacrylamide gel electrophoresis component with an apparent molecular weight of 55 000 is particularly enhanced. Moreover, concanavalin A induces excretion of this component into the cultivation medium. 6. 6. The data are discussed in terms of possible modes of interaction between concanavalin A and thymocyte plasma membranes.