Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

scholarly journals Corynebacterium diphtheriae: Identification and Characterization of a Channel-Forming Protein in the Cell Wall

2007 ◽  
Vol 189 (21) ◽  
pp. 7709-7719 ◽  
Author(s):  
Bettina Schiffler ◽  
Enrico Barth ◽  
Mamadou Daffé ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Single Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Corynebacterium Diphtheriae ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lipid Bilayer Membranes ◽  
Whole Cells

ABSTRACT The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8r(−) Tox− (= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis

1989 ◽  
Vol 35 (8) ◽  
pp. 749-753 ◽  
Author(s):  
Denis Leclerc ◽  
Alain Asselin
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Gel Electrophoresis ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cell Wall Hydrolases

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).Key words: autolysins, electrophoresis, hydrolases, muramidases, peptidoglycan.

scholarly journals Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein fromRhodococcus erythropolis

2000 ◽  
Vol 182 (3) ◽  
pp. 764-770 ◽  
Author(s):  
Thomas Lichtinger ◽  
Gila Reiss ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Single Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Sequencing ◽  
Cell Wall Fraction ◽  
Channel Conductance ◽  
Single Channel Conductance

ABSTRACT Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel ofR. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.

scholarly journals Deletion of New Covalently Linked Cell Wall Glycoproteins Alters the Electrophoretic Mobility of Phosphorylated Wall Components of Saccharomyces cerevisiae

1999 ◽  
Vol 181 (10) ◽  
pp. 3076-3086 ◽  
Author(s):  
Vladimir Mrsa ◽  
Margit Ecker ◽  
Sabine Strahl-Bolsinger ◽  
Manfred Nimtz ◽  
Ludwig Lehle ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Radioactive Material ◽  
Cell Wall Proteins ◽  
Triple Mutant ◽  
Calcofluor White ◽  
Covalently Linked

ABSTRACT The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied.33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N -glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368–3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154–2162, 1997). In ccw12, ccw13, orccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, theccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.

scholarly journals Fractionation of the proteins of plant microbodies

1974 ◽  
Vol 144 (3) ◽  
pp. 559-566 ◽  
Author(s):  
R H Brown ◽  
J M Lord ◽  
M J Merrett
Keyword(s):  
Membrane Proteins ◽  
Ricinus Communis ◽  
Osmotic Shock ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Weight One ◽  
Protein Components

1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ‘ghost’ proteins). 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of ‘ghost’ fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000–28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ‘ghosts’ indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).

scholarly journals The quaternary structure of wheat-germ aspartate transcarbamoylase

1982 ◽  
Vol 203 (2) ◽  
pp. 413-417 ◽  
Author(s):  
R J Yon ◽  
J E Grayson ◽  
A Chawda ◽  
P J Butterworth
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Quaternary Structure ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Aspartate Transcarbamoylase ◽  
Molecular Masses

1. The molecular mass of aspartate transcarbamoylase purified from wheat germ was found to be 101kDa by sucrose-density-gradient centrifugation, 103kDa by gel-filtration chromatography and 108kDa by polyacrylamide-gel electrophoresis. A mean value of 104 +/- 11kDa was obtained by pooling several replicate results from each method. 2. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated a single size of polypeptide chain of mean molecular mass 37 +/- 4kDa. The ratio of the mean molecular masses of the active and denatured enzymes is 2.8.3. When the active enzyme was covalently cross-linked at a low protein concentration by dimethyl suberimidate, and then examined electrophoretically under denaturing conditions, three size species were observed to predominate, of apparent molecular masses 36, 77 and 106kDa respectively. 4. These results indicate that the intact, fully regulatory enzyme is a simple trimer, slightly larger than the trimeric ‘catalytic subunit’ of the aspartate transcarbamoylase from Escherichia coli [Weber (1968) Nature (London) 218, 1116-1118]. The prevalence of trimeric structures amongst carbamoyl-transferase enzymes is discussed.

scholarly journals Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

1985 ◽  
Vol 228 (1) ◽  
pp. 111-117 ◽  
Author(s):  
E Davies Jones ◽  
F A Hashim ◽  
Y Kajita ◽  
F M Creagh ◽  
P R Buckland ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyrotropin Receptor ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

scholarly journals Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

2009 ◽  
Vol 75 (14) ◽  
pp. 4661-4667 ◽  
Author(s):  
Alejandro Hernández-Soto ◽  
M. Cristina Del Rincón-Castro ◽  
Ana M. Espinoza ◽  
Jorge E. Ibarra
Keyword(s):  
Bacillus Thuringiensis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Microbial Control ◽  
Control Agent ◽  
Mass Spectrometry Analysis ◽  
Parasporal Crystal ◽  
Spectrometry Analysis ◽  
Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

2009 ◽  
Vol 72 (12) ◽  
pp. 2524-2529 ◽  
Author(s):  
JINLAN ZHANG ◽  
GUORONG LIU ◽  
NAN SHANG ◽  
WANPENG CHENG ◽  
SHANGWU CHEN ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Gel Chromatography ◽  
Original Activity ◽  
Lactobacillus Pentosus ◽  
Spectrometry Analysis

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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