Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation

2019 ◽  
pp. 299-310
Author(s):  
Ricardo F. dos Santos ◽  
Cátia Bárria ◽  
Cecília M. Arraiano ◽  
José M. Andrade
Keyword(s):  
Sucrose Gradient ◽  
Sucrose Gradient Ultracentrifugation

scholarly journals Correction to: Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation

2020 ◽  
pp. C1-C1
Author(s):  
Ricardo F. dos Santos ◽  
Cátia Bárria ◽  
Cecília M. Arraiano ◽  
José M. Andrade
Keyword(s):  
Sucrose Gradient ◽  
Sucrose Gradient Ultracentrifugation

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
David R Graham ◽  
Antoine Younes ◽  
Alexey Lyashkov ◽  
Anna Sheydina ◽  
Maria Volkova ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lipid Domains ◽  
Rt Pcr ◽  
Pacemaker Cells ◽  
Pacemaker Function ◽  
Sucrose Gradient Ultracentrifugation

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.

scholarly journals Structural features of the first component of human complement, C1, as revealed by surface iodination

1982 ◽  
Vol 203 (1) ◽  
pp. 185-191 ◽  
Author(s):  
C L Villiers ◽  
S Chesne ◽  
M B Lacroix ◽  
G J Arlaud ◽  
M G Colomb
Keyword(s):  
Sucrose Gradient ◽  
Structural Features ◽  
Complement Component ◽  
Monomeric Form ◽  
Human Complement ◽  
Equal Distribution ◽  
A Chain ◽  
Sucrose Gradient Ultracentrifugation ◽  
Label Distribution ◽  
Human Complement Component

Lactoperoxidase-catalysed surface iodination and sucrose-gradient ultracentrifugation were used to investigate the structure of human complement component C1. 1. Proenzymic subcomponents C1r and C1s associated to form a trimeric C1r2-C1s complex (7.6 S) in the presence of EDTA, and a tetrameric Clr2-C1s2 complex (9.1 S) in the presence of Ca2+. Iodination of the 9.1 S complex led to a predominant labelling of C1r (70%) over C1s (30%), essentially located in the b-chain moiety of C1r and in the a-chain moiety of C1s. 2. Reconstruction of proenzymic soluble C1 (15.2 S) from C1q, C1r and C1s was partially inhibited when C1s labelled in its monomeric form was used and almost abolished when iodinated C1r was used. Reconstruction of fully activated C1 was not possible, whereas hybrid C1q-C1r2-C1s2 complex was obtained. 3. Iodination of proenzymic or activated C1 bound to IgG-ovalbumin aggregates led to an equal distribution of the radioactivity between C1q and C1r2-C1s2. With regard to C1q, the label distribution between the three chains was similar whether C1 was in its proenzymic or activated form. Label distribution in the C1r2-C1s2 moiety of C1 was the same as that obtained for isolated C1r2-C1s2, and this was also true for the corresponding activated components. However, two different labelling patterns were found, corresponding to the proenzyme and the activated states.

Fractionation and antigenic characterization of organelles ofEimeria tenellasporozoites

Parasitology ◽  
1992 ◽  
Vol 104 (1) ◽  
pp. 1-9 ◽  
Author(s):  
U. Kawazoe ◽  
F. M. Tomley ◽  
J. A. Frazier
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Sucrose Gradient ◽  
Eimeria Tenella ◽  
2Nd Generation ◽  
Antigenic Characterization ◽  
Electron Microscopical ◽  
Sucrose Gradient Ultracentrifugation ◽  
Antigenic Reactivity

SUMMARYSporozoites ofEimeria tenellawere disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.

scholarly journals Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

2021 ◽  
Author(s):  
RB Gorodnichev ◽  
MA Kornienko ◽  
NS Kuptsov ◽  
AD Efimov ◽  
VI Bogdan ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sucrose Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Phage Lysate ◽  
Purification Method ◽  
Cscl Density Gradient ◽  
Cell Components ◽  
Purification Methods ◽  
Sucrose Gradient Ultracentrifugation

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

scholarly journals Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell

1998 ◽  
Vol 333 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Toshio TOMITA ◽  
Dai ISHIKAWA ◽  
Takayasu NOGUCHI ◽  
Eisaku KATAYAMA ◽  
Yohichi HASHIMOTO
Keyword(s):  
Sucrose Gradient ◽  
Edible Mushroom ◽  
Human Erythrocytes ◽  
Flammulina Velutipes ◽  
Target Cells ◽  
Potassium Ions ◽  
Fruiting Bodies ◽  
Sucrose Gradient Ultracentrifugation ◽  
Ring Shaped Structure ◽  
Cytolytic Action

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of > 5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 °C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 °C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4–5 nm on target cells.

CONGENITAL GOITRE IN SHEEP: ISOLATION OF THE IODOPROTEINS WHICH REPLACE THYROGLOBULIN

1976 ◽  
Vol 71 (2) ◽  
pp. 179-192 ◽  
Author(s):  
C. E. DOLLING ◽  
B. F. GOOD
Keyword(s):  
Molecular Weight ◽  
Thyroid Gland ◽  
Sucrose Gradient ◽  
Sedimentation Coefficient ◽  
Agarose Gel ◽  
Merino Sheep ◽  
Thyroid Glands ◽  
Sucrose Gradient Ultracentrifugation ◽  
Australian Merino

SUMMARY Immunological and chromatographic studies of proteins from the congenital goitre of South Australian Merino sheep revealed that normal thyroglobulin is absent from the thyroid glands of these sheep. However, a thyroglobulin-immunoreactive iodoprotein was isolated by affinity chromatography on agarose gel to which antibody against thyroglobulin had been covalently bound. Sucrose-gradient ultracentrifugation indicated that this iodoprotein had a sedimentation coefficient of 8S and a molecular weight of approximately 175000. This iodoprotein is therefore about one quarter the size of normal thyroglobulin (19S; 660000) and is similar in size to the subunit of thyroglobulin (3-8S; 165000) although this has usually been described as non-iodinated except when derived by reductive fission. In addition the goitre extract contained iodoproteins which had the immunological properties of serum albumin and immunoglobulin G. Determination of the iodine and iodoamino acid content of the hydrolysed iodoproteins revealed that they contained iodothyronines which were able to contribute to the production of thyroid hormones although the total iodothyronine content of the goitrous gland was less than that of the normal sheep thyroid gland.

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