Comparison of cell wall and membrane proteins from eight Candida species

Cell surface proteins from eight Candida species were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell wall proteins of these species were similar in their electrophoretic patterns. On the other hand, differences were detected among the patterns of membrane proteins, although C. albicans serotypes A and B gave similar electrophoretic patterns, and so did two species (C. pseudotropicalis and C. parapsilosis).     

Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus

Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.     

Isolation and immunochemical characteristics of the immunodepressive substance from Escherichia coli.

A substance, inhibiting the production of haemolysins against sheep erythrocytes in mice was isolated from two non-pathogenic strains of E. coli, 020:K4 and M-17, by the methods of differential centrifugation and gel filtration. Spectrophotometric studies and chemical analysis have shown the isolated substance to be glycolipid. The immunodepressive substance is localized in the cytoplasm of the microbial cell. The isolated and partly purified immunodepressive substance did not contain any admixture of O-antigen (endotoxin) of the cell wall or of antigens giving cross reactions with sheep erythrocytes. The isolated substance exhibited weak antigenic properties and was not toxic for mice when administered in a dose of 2 mg (dry weight).     

Ultrastructure of the Corynebacterium glutamicum cell wall

The cell wall structure of the Gram-positive Corynebacterium glutamicum was evaluated by electron microscopy of thin sections after freeze-substitution and conventional fixation with glutaraldehyde. For the cell wall an overall thickness of approximately 32 nm was determined, with 8.5 nm corresponding to an outer layer, 6.5 nm to an electron translucent region (ETR) as found in mycobacteria and 17 nm to the peptidoglycan. Knob-like surface structures previously observed in freeze-fracture experiments were detected when cells were conventionally processed with a fixation using glutaraldehyde. By mild treatment with detergents approximately 20 proteins were extracted from the cell wall. From seven of these N-terminal amino acid sequences were determined.     

Study of membrane proteins from Microccus lysodeikticus using immunochemical methods]

Using immunoelectrophoresis, the antigenicity of various protein fractions of the Micrococcus lysodeikticus membranes was evaluated. It was shown that both the peripheral and integral membrane proteins possess the antigenic determinants. The antiserum exhausted by the M. lysodeikticus mebranes loses its ability to interact with intergral proteins, which are not solubilized by Triton X-100. It was thus assumed that the integral proteins are exposed on the membrane surface constantly or periodically and that there exist no proteins which are completely and permanently incorporated into the lipid bilayer. The respiratory chain of the M. lysodeikticus membrane is inhibited by membrane immunoglobulins by 50%. This is probably due to the presence in the membrane antiserum of antibodies specific to the respiratory chain enzymes. Evidence for this assumption can be derived from the fact that partially purified cytochrome b556 forms a precipitation zone with the membrane antiserum and that the activity of membrane NADH-dehydrogenase is inhibited by a monoserum against NADH-dehydrogenase.     

Isolation and characteristics of insulin-binding proteins from soybeans

Two soybean insulin-binding proteins were isolated using affinity chromatography on insulin-Sepharose. Both proteins have molecular mass about 39 kDa and consist of two subunits linked by disulphide bonds. According to the amino acid composition and N-terminal sequences of the subunits, these proteins, characterized by the absence of free thiol groups and sugar residues, are variants of the previously described soybean basic 7S globulin. The blotted proteins as well as their subunits were shown to bind 125I-labelled bovine insulin. For one of the proteins and insulin, dissociation constant of 4.10(-9) M was measured. The existence of plant insulin-binding proteins suggests the insulin-like regulation in the plant metabolism.     

Isolation and characteristics of DNA fragments bound to mitochondrial membrane proteins

Fragments of rat liver mitochondrial DNA were isolated. In vivo these fragments were able to form the complexes with the proteins of inner mitochondrial membrane. The fragments represent unique DNA regions with the secondary structure, their A-T content being equal to 82%. With the aid of phosphomonoesterase, polynucleotidkinase and gamma-(32P)-ATP mtDNA fragments were labeled and analyzed for oligopyrimidine composition. It was shown that they were enriched in di- and tri-oligo-pyrimidine blocks. The fragments are shown to form in vitro a complex with the membrane proteins. A single protein m. wt. 40,000) was reisolated from the complex.     

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.     

Isolation and partial characterization of soluble alfalfa proteins

Soluble proteins, representing ⋍18% of total plant protein, were extracted from freshly harvested alfalfa. Two fractions based on solubility in one-half saturated (NH₄)₂SO₄—Fraction G (insoluble) and Fraction A (soluble)—were obtained, representing two-thirds and one-third of the extracted protein, respectively. Fraction G contained less hexose-like and more lipid substances than Fraction A which was characterized by an unusually high ash content. Gel electrophoretic patterns revealed greater heterogeneity in Fraction A than in Fraction G, whereas the reverse situation was evident in sedimentation-velocity patterns. The three sedimenting boundaries apparent in Fraction G had S_20,app values of 29.2 (4%), 17.4 (61%) and 3.2 (35%). The S_20,app 3.2 boundary corresponded to the single boundary observed for Fraction A. Moving-boundary electrophoresis revealed a single boundary for each fraction, possessing mobilities of −6.5 (Fraction G) and −7.5 (Fraction A) Tiselius units, respectively.     

Isolation and characteristics of the plasma membrane fraction from the swine myometrium

An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles.     

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.     

Isolation and characteristics of surface proteins from Streptococcus group A

Cell wall surface proteins of group A Streptococcus type 29 were extracted with 1 M hydroxylamine pH 6.0. The purification procedure included fractionation with ammonium sulfate and gel filtration on Sephadex G-150. SDS polyacrylamide gel electrophoresis revealed a number of proteins (approximately 20) with molecular mass of 70 kD; the difference in Mr between the proteins was 5-10 kD. Isoelectrofocusing demonstrated that the proteins are either acid (pI = 3.7) or weakly alkaline (pI = 7.7). Possible reasons for the heterogeneity of Streptococcus cell wall surface proteins are discussed.