Studies on the cell wall of Spirillum serpens. II. Chemical characterization of the outer structured layer

1973 ◽  
Vol 19 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Francis L. A. Buckmire ◽  
Robert G. E. Murray
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Self Assembly ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Guanidine Hydrochloride ◽  
Chemical Characterization ◽  
Wall Layer ◽  
Outer Cell ◽  
Density Gradients

The outer cell-wall layer of Spirillum serpens VHA, composed of a hexagonal array of macromolecules, was dissociated from 'cleaned' cell-wall fragments with 1.5 M guanidine hydrochloride, pH 7.0. The soluble material contained 98% protein, 2% carbohydrate, and no ethanolamine, phosphate, RNA, or DNA. Evidence for the homogeneity of the isolated cell-wall protein was obtained by sedimentation velocity, polyacrylamide disc gel electrophoresis, molecular sieve chromatography on Sephadex G-200, and sucrose density gradients. The protein was acidic and had a minimum molecular weight of about 48 000 daltons calculated from the amino acid analysis. A molecular weight of between 125 000 and 150 000 daltons was obtained for the protein by polyacrylamide disc-gel electrophoresis, sucrose density gradients, and molecular sieve chromatography. The sedimentation rate of the protein was considerably less in 1.5 M guanidine hydrochloride, pH 7.0, than in its absence although the protein remained homogenous under both conditions. Self-assembly of the purified protein into its original hexagonal layer on a template of wall fragments from which the protein had been removed previously was obtained when the mixture was incubated in the presence, but not in the absence of Ca2+. It seems likely that the particles (hexagons) seen on the surface of the cell are an assembly of trimers.

The isolation of surface array proteins from bacteria

1984 ◽  
Vol 62 (11) ◽  
pp. 1181-1189 ◽  
Author(s):  
S. F. Koval ◽  
R. G. E. Murray
Keyword(s):  
Molecular Weight ◽  
Protein Conformation ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Features ◽  
Proteolytic Degradation ◽  
Low Concentrations ◽  
Single Polypeptide

The methods used for the isolation of regularly structured (RS) surface array proteins of a range of prokaryotes are described. Most RS proteins can be selectively solubilized from envelope preparations with low concentrations of urea or guanidine hydrochloride. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis of the protein extracts shows that most RS arrays are composed of a single polypeptide that may contain carbohydrate. The molecular weight of the proteins varies from 41 000 to 200 000. Possible reasons for the presence of more than one polypeptide in RS protein preparations are discussed, as well as the evidence for proteolytic degradation of some RS proteins during isolation. Structural features of the RS proteins are described and the importance of protein conformation to assembly of the arrays is indicated.

scholarly journals Conidial Hydrophobins of Aspergillus fumigatus

2003 ◽  
Vol 69 (3) ◽  
pp. 1581-1588 ◽  
Author(s):  
Sophie Paris ◽  
Jean-Paul Debeaupuis ◽  
Reto Crameri ◽  
Marilyn Carey ◽  
Franck Charlès ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Amorphous Layer ◽  
Wall Layer ◽  
Host Cells ◽  
Outer Cell ◽  
Host Immune System ◽  
Wild Type ◽  
Cell Wall Layer ◽  
Outer Cell Wall

ABSTRACT The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and ΔrodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. ΔrodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of ΔrodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the ΔrodA ΔrodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.

Studies on the cell wall of Spirillum serpens. 1. Isolation and partial purification of the outermost cell wall layer

1970 ◽  
Vol 16 (10) ◽  
pp. 1011-1022 ◽  
Author(s):  
Francis L. A. Buckmire ◽  
Robert G. E. Murray
Keyword(s):  
Cell Wall ◽  
Intact Cell ◽  
Guanidine Hydrochloride ◽  
Casein Hydrolysate ◽  
Wall Layer ◽  
Partial Purification ◽  
Repeated Heating ◽  
Physical Study ◽  
Backing Layer ◽  
Wall Components

The presence of the hexagonal array of macromolecules on the outer surface of the cell wall of Spirillum serpens VHA required the addition of calcium to an otherwise effective growth medium (vitamin-free casein hydrolysate); slightly improved growth resulted from addition of a complex salts mixture. A means of isolating this layer for chemical and physical study was sought and controlled by electron microscopy of freeze-etched, negatively stained, and sectioned preparations. The structure was destroyed, extracted, or removed by extremes of pH (<4.5, >9), 1 M guanidine hydrochloride (pH 7), 2 M urea, and dimethyl sulfoxide, with varying damage to the cell. Heat (60° for 1 h) removed much of the outer layer from the intact cell as an array of units disposed on a delicate backing layer, leaving the basic Gram-negative triplet wall components. These fragments remained stable through washing and repeated heating in the presence of 0.001 M calcium chloride. Guanidine hydrochloride (1.5 M) dissolved the units from the tubes and vesicles formed by the backing layer. Dialysis against water removed salts and guanidine, caused the precipitation of residual contaminants, and provided a supernatant which, when lyophilized, provided a product containing 98% protein.

scholarly journals Influence of the Secondary Cell Wall Polymer on the Reassembly, Recrystallization, and Stability Properties of the S-Layer Protein from Bacillus stearothermophilus PV72/p2

1998 ◽  
Vol 180 (16) ◽  
pp. 4146-4153 ◽  
Author(s):  
Margit Sára ◽  
Christine Dekitsch ◽  
Harald F. Mayer ◽  
Eva M. Egelseer ◽  
Uwe B. Sleytr
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Secondary Cell Wall ◽  
Bacillus Stearothermophilus ◽  
Guanidine Hydrochloride ◽  
Terminal Region ◽  
Water Soluble ◽  
Outer Face ◽  
Cell Wall Polymer ◽  
Secondary Cell Wall Polymer

ABSTRACT The high-molecular-weight secondary cell wall polymer (SCWP) fromBacillus stearothermophilus PV72/p2 is mainly composed ofN-acetylglucosamine (GlcNAc) andN-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 μg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-l-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.

Soluble Crosslinked Fibrin(ogen) Polymers

1985 ◽  
Vol 54 (04) ◽  
pp. 804-807 ◽  
Author(s):  
Eberhard Selmayr ◽  
Gert Müller-Berghaus
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Molecular Size ◽  
Density Gradients ◽  
Reducing Conditions ◽  
Diagnostic Criterion

SummaryThe present study is concerned with the formation of fibrin-fibrinogen associations in the presence of FXIIIa while fibrinolysis was inhibited by aprotinin and EACA. SDS agarose-poly acryl-amide gel electrophoresis on 2.5% gels under non-reducing conditions and ultracentrifugation of the associations on urea-sucrose density gradients showed the formation of soluble crosslinked high molecular weight (HMW) fibrin-fibrinogen polymers with an estimated molecular size up to 10 times that of fibrinogen. After incubation of a mixture of 131I-fibrinogen (4 mg/ml) and 125I-desAA-fibrin (0.2 mg/ml) with FXIIIa (2 U/ml) for 1 h at 37ΰ C, about 5% of the fibrinogen and 80% of the fibrin was incorporated into the generated soluble HMW polymers. The detection of soluble crosslinked fibrin-fibrinogen polymers could be a useful diagnostic criterion for imminent DIC.

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase

1977 ◽  
Vol 55 (1) ◽  
pp. 1-8 ◽  
Author(s):  
D. J. Mahuran ◽  
Ronald H. Angus ◽  
Carl V. Braun ◽  
S. S. Sim ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sulfhydryl Groups ◽  
Ph Optimum ◽  
Amino Terminal ◽  
Diketo Acids ◽  
Hydrolysis Of

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 ± 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 – 43 000, as determined by gel electrophoresis, gel filtration in guanidine–hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine–hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent. The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported.The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Planta ◽  
1981 ◽  
Vol 151 (3) ◽  
pp. 247-250 ◽  
Author(s):  
J. Burczyk ◽  
H. Szkawran ◽  
I. Zontek ◽  
F. -Ch. Czygan
Keyword(s):  
Cell Wall ◽  
Wall Layer ◽  
Outer Cell ◽  
Cell Wall Layer ◽  
Outer Cell Wall

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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