scholarly journals Reconstitution of an Allophycocyanin Trimer Complex Containing the C-Terminal 21-23 kDa Domain of the Core-Membrane Linker Polypeptide Lcm

1994 ◽  
Vol 49 (5-6) ◽  
pp. 331-336 ◽  
Author(s):  
Lothar Gottschalk ◽  
Friedrich Lottspeich ◽  
Hugo Scheer
Keyword(s):  
Density Gradient ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Preparative Scale ◽  
Small Core ◽  
The Core ◽  
Spectral Changes ◽  
Sds Page ◽  
Molecular Masses

Abstract Allophycocyanin (AP) was isolated from extracts of the cyanobacterium Mastigocladus laminosus. A fraction enriched in AP-associated polypeptides with apparent molecular masses of 21 -23 kD a in SDS-PAGE, was isolated on a preparative scale and identified as a homolo­gous mixture of C-terminal fragments of the core-membrane linker polypeptide Lcm. The complex (αAPβAP)3 ·21 -23 kD a was reconstituted and characterized by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. The 21 -23 kD a polypeptides were found to induce spectral changes in AP similar to those induced by the small core linker polypeptide Lc8.9. Possible functions of the complex in phycobilisomes are discussed.

scholarly journals Protein aggregation. Studies of larger aggregates of C-phycocyanin

1968 ◽  
Vol 110 (3) ◽  
pp. 457-464 ◽  
Author(s):  
J. J. Lee ◽  
D. S. Berns
Keyword(s):  
Density Gradient ◽  
Size Range ◽  
Gel Filtration ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Denatured Protein ◽  
Elution Pattern ◽  
Green Algal ◽  
Fluorescence Efficiency ◽  
Algal Extracts

Aggregates of phycocyanin sedimenting at 17s, 22s and 27s are demonstrated to constitute more than 40% of crude blue–green-algal extracts, pH6·0 and I0·1, and are retained in highly purified preparations. Sedimentation-velocity studies of the large aggregates as a function of pH are reported. Sucrose-density-gradient experiments performed as a function of time of sedimentation indicate that: (1) with increasing time of sedimentation, the largest aggregates are dissipated at the leading protein boundary and the several phycocyanin species present are not completely resolved; (2) phycocyanin fractions with the largest aggregates exhibit the highest E620/E280 ratio and the largest relative fluorescence efficiency. Gel-filtration experiments with Sephadex G-200 do not resolve the species completely, and reapplication of phycocyanin gel-filtration fractions to the column results in an elution pattern similar to the original, except that there is an enhancement of the allophycocyanin fraction and the amount of denatured protein. Increasing the sedimentation times in a sucrose density gradient also enhances the allophycocyanin fraction. Fluorescence results demonstrate that there are possibly three excitation maxima, one corresponding to the monomer (approx. 600mμ), one for higher aggregates (625–630mμ) and one for the allophycocyanin fraction (approx. 650mμ). Only a single fluorescence-emission band is detected, which is fairly symmetrical and which has a red shift with higher aggregation and with the appearance of allophycocyanin. The appearance of allophycocyanin may be correlated with the irreversible disaggregation of the largest phycocyanin species. It is suggested that the largest protein aggregates are in the size range of the biliprotein aggregates reported in electron microscopy of algal cells.

scholarly journals THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

1964 ◽  
Vol 120 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Burton R. Andersen ◽  
Wilton E. Vannier
Keyword(s):  
Density Gradient ◽  
Sedimentation Rate ◽  
Gradient Method ◽  
Sucrose Density Gradient ◽  
Major Portion ◽  
Density Gradient Ultracentrifugation ◽  
Cell Method ◽  
Average Value ◽  
Γ Globulins

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

scholarly journals Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin

1968 ◽  
Vol 110 (3) ◽  
pp. 465-470 ◽  
Author(s):  
J. J. Lee ◽  
D. S. Berns
Keyword(s):  
Density Gradient ◽  
Deuterium Oxide ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Structural Protein ◽  
Visible Absorption ◽  
Large Size ◽  
Fluorescence Efficiency ◽  
Emission Studies

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5·5 to 7·0. Sucrose-density-gradient studies in deuterium oxide at pD6·0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E620/E280 ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.

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 Hypoprothrombinemia: case report

Blood ◽  
1978 ◽  
Vol 51 (2) ◽  
pp. 299-306
Author(s):  
RR Montgomery ◽  
A Otsuka ◽  
WE Hathaway
Keyword(s):  
Case Report ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Factor Ii ◽  
History Of

A patient with a significant history of spontaneous and posttraumatic bleeding was found to have hypoprothrombinemia. His prothrombin (factor II) activity by clotting assay was 9.5% and his factor II antigen was 5%. Crossed immunoelectrophoresis and sucrose density gradient ultracentrifugation of the patient's plasma showed his prothrombin to be qualitatively indistinguishable from normal prothrombin by these techniques.

scholarly journals Oral toxicities of Clostridium botulinum type C and D toxins of different molecular sizes

1980 ◽  
Vol 28 (2) ◽  
pp. 303-309
Author(s):  
I Ohishi ◽  
G Sakaguchi
Keyword(s):  
Density Gradient ◽  
Clostridium Botulinum ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Botulinum Toxins ◽  
Molecular Dissociation ◽  
Botulinum Type ◽  
Molecular Sizes ◽  
Type C ◽  
Progenitor Toxin

Clostridium botulinum type C progenitor toxins of different molecule sizes, C-L (16S) and C-M (12S), were purified from cultures of strains 573, Stockholm, and CB-19. C-L toxin showed some hemaggglutinin activity, whereas C-M toxin did not. Neither C-L nor C-M toxin was activated upon trypsinization. Molecular dissociation of purified type C-L and C-M toxins into toxic and nontoxic components was demonstrated by sucrose density gradient ultracentrifugation and diethylaminoethyl-Sephadex chromatography at pH 8.0. The molecular construction of type C progenitor toxin appears to be analogous to that reported for botulinum toxins of other types. C-L and D-L toxins showed higher oral toxicities to mice than did C-M or D-M toxin. Such higher oral toxicities were ascribed to the higher stabilities of these toxins in gastric and intestinal juices.

scholarly journals CHARACTERIZATION OF IMMUNOGLOBULIN STRUCTURES FROM THE SURFACE OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

1971 ◽  
Vol 134 (1) ◽  
pp. 265-280 ◽  
Author(s):  
Trond Eskeland ◽  
Eva Klein ◽  
Masaharu Inoue ◽  
Bo Johansson
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Density Gradient Ultracentrifugation ◽  
Freezing And Thawing ◽  
Passive Hemagglutination

Chronic lymphocytic leukemia cells with relatively large amounts of mu and kappa immunoglobulin structures on the surface, and apparently very small amounts intracellularly, were subjected to homogenization or washing after freezing and thawing. After a light centrifugation, which sedimented the nuclei and unbroken cells, most of the immunoglobulin structures were found in the supernatant. Ultracentrifugation, which was performed to remove the membranes from the supernatant, sedimented only half the amount of the immunoglobulin structures. By sucrose density gradient ultracentrifugation and Sephadex G-200 filtration, the unsedimented immunoglobulin structures were shown to consist of 7S IgM and free kappa chains. About 80,000 7S IgM molecules were calculated to be present on each cell. The amount of kappa chains not associated with IgM was estimated to be equal to the amount of kappa chains in IgM. Inhibition of passive hemagglutination was used to detect and quantitate the immunoglobulin structures.

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