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At least 343 records · Page 19Linked to original sources
PubMed1979

The use of equilibrium density-gradient ultracentrifugation in the isolation and characterisation of glycoproteins with blood group P1 activity from sheep hydatid-cyst fluid.

Equilibrium density-gradient ultracentrifugation in caesium choride and caesium sulphate has been used in the isolation and fractionation of the glycoproteins specific for blood-group P1 from hydatid cyst fluids. The fractions obtained have distinct and systematic differences specifically related to their buoyant densities, chemical compositions and specific-activities for group P1. High levels of specific-activity were maintained over a large range of chemical compositions. The peptide content varied systematically from 2.5% for the densest fraction to 37% for the least dense fraction. The amino acid composition was essentially constant over all fractions. The proportion of glucosamine decreased and the proportions of galactosamine, mannose and glucose increased with increasing peptide content of the fractions. The data presented suggest the present of oligosaccharide side-chains of various lengths and compositions and/or the presence of oligosaccharide side-chains with very different chemical compositions, of which only some are associated with the specificity for group P1. The properties of the glycoproteins from hydatid cyst fluids have been compared with those of the glycoproteins from human ovarian cysts. Although some similarities have been demonstrated there are significant differences.

Amino Acids↗
PubMed1988

Visualization of reversible macromolecular reactions in an analytical ultracentrifuge.

It has been shown that the saw-like disturbances of sedimentation observed in an analytical ultracentrifuge are not caused by convective disturbances of the solution but result from a special type of intermolecular reaction of reversible association/dissociation. A qualitative theory of saw-like anomalies has been suggested and the sedimentation and kinetic conditions of their origin have been indicated. Such reactions are a frequent occurrence in the serum of patients affected with rheumatic diseases and acute myocardial infarction. Experimental data indicate the involvement of immunoglobulins (viz., low-affinity antibodies) which form reversible immune complexes. Saw-like sedimentation patterns, especially those of the schlieren type, are a direct testimony to reversible association/dissociation reactions in macromolecular solutions, whereas other experimental methods provide only oblique evidence.

Antibody Affinity↗
PubMed1996

Associations between erythrocyte band 3 protein and aldolase in detergent solution. Determining their stoichiometry by analytical ultracentrifugation.

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.

Animals↗
PubMed1997

An ultracentrifugal approach to quantitative characterization of the molecular assembly of a physiological electron-transfer complex: the interaction of electron-transferring flavoprotein with trimethylamine dehydrogenase.

The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4 degrees C. Analysis of sedimentation-equilibrium distributions obtained at 15 000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119-130] has yielded an intrinsic dissociation constant of 3-7 microM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.

Bacteria↗
PubMed1966

Influenza virus purification with the zonal ultracentrifuge.

Sufficient amounts of influenza virus (density, 1.185; size, 722S) can be highly purified (22,000 chicken cell agglutinating units per milligram of protein) with a zonal ultracentrifuge, used first in a rate process followed by isopycnic banding, to permit its detailed biological and physical-chemical evaluation.

Animals↗
PubMed1967

Cytoplasmic and chloroplast ribosomes of Chlamydomonas: ultracentrifugal characterization.

Ribosomes isolated from the cytoplasmic and chloroplast fractions of Chlamydomonas were characterized in the ultracentrifuge. The cytoplasmic ribosomes belong to the 80S class of ribosomes, and, like animal ribosomes, dissociate to 60, 50, and 40S subunits. However, like the ribosomes of microorganisms, they contain smaller RNA's, 24 and 16S, and require 0.01 mole of magnesium ions per liter for stability. Chloroplast ribosomes are 70S like those of higher plants but are very unstable. A stable 50S subunit has been observed.

Chloroplasts↗
PubMed1972

Preparation of purified suspensions of Coxiella burneti by Genetron extraction followed by continuous-flow ultracentrifugation.

A method for the preparation of purified suspensions of Coxiella burneti by Genetron extraction followed by continuous-flow density gradient ultracentrifugation is described. Both phases of the Henzerling strain of C. burneti were found in a zone between 53 and 65% (w/w) sucrose. Based on chemical assays, the Genetron zonal rickettsial suspensions were found to be as pure as the rickettsial suspensions which were prepared by the ether extraction method currently in use for producing Q fever vaccines for human use.

Animals↗
PubMed1971

Ultracentrifugal analysis of staphylococcal alpha toxin.

Ultracentrifugal examination of staphylococcal alpha toxin at different stages of purification showed the presence of a major component having a sedimentation coefficient of 2.8S, present to the extent of more than 90% of the sample, and identifiable with active toxin. Several minor components having S(20,w) values of 11.5S, 8.5S, and 2.0S were detected. The 11.5S component presumably is identical with a toxin aggregate studied earlier and designated 12S; the 8.5S component appears to be delta toxin. A sedimentation equilibrium study of more highly purified material gave 32,700 as the best estimate of molecular weight of alpha toxin. Lowering the pH of the partially purified alpha toxin from 10.2 to 5.3 resulted in a small increase in S(20,w) of the 11.5S component and in the disappearance of the 8.5S component, whereas the S(20,w), molecular weight, and hemolytic activity of the toxin remained constant. Exposure of toxin to pH 3.5 irreversibly reduced the S(20,w) to 2.0S, the molecular weight to about 16,000, and caused irreversible inactivation. Raising the pH of acid-inactivated toxin and adding sodium dodecyl sulfate to 1% increased the S(20,w) to near its normal value (2.7S) but did not restore activity.

Borates↗
PubMed1982

Purification of Pasteurella multocida antigens by ultracentrifugation and isoelectrofocusing.

A procedure was developed to purify Pasteurella multocida X-731 antigens extracted by potassium thiocyanate. The crude extract was centrifuged at 105 000 x g; the antigens were then separated into a particulate (40p) fraction and a soluble (40s) fraction consisting of proteins and polysaccharides. These fractions were antigenically different. The ultracentrifuged antigens were resolved further by preparative isoelectrofocusing. The 40p antigens focused in a pH range of 3.0 to 6.0; distinctive proteins focused at pH's of 3.5, 3.6, and 3.8. The electrofocused 40p antigens were antigenically similar. The 40s antigens were initially electrofocused in a broad pH range and were found within a pH range of 4.6 to 9.0. The process was repeated with a narrower pH range and antigens that were focused in a narrower pH range could be separated and unique antigenic activities identified. Specific antigens from defined pH ranges were pooled and examined further by immunoelectrophoresis, analytical electrofocusing, and sodium dodecyl sulphate--polyacrylamide gel electrophoresis.

Antigens, Bacterial↗
PubMed1969

Chromatographic, ultracentrifugal, and related studies of fibrinogen "Baltimore".

Chromatographic, ultracentrifugal, and related studies of the fibrinogen of a patient with a congenital disorder of fibrinogen (fibrinogen "Baltimore" have provided evidence of structural differences from normal.Diethylaminoethyl-cellulose (DEAE-cellulose) gradient elution chromatography demonstrated two major peaks in the elution pattern of fibrinogen Baltimore as was the case for normal fibrinogen. However, the first peak of fibrinogen Baltimore was somewhat broader and more symmetrical and was eluted significantly later in the chromatogram than the corresponding peak of normal fibrinogen. Additionally, in some elution patterns, a shoulder on the ascending limb of peak 1 was present, suggesting the presence of chromatographically "normal" fibrinogen. Thrombin time determinations of eluted column fractions from a chromatogram of propositus fibrinogen supported this conclusion by demonstrating that fibrinogen from the ascending portion of peak 1 behaved functionally more like normal than that later in the chromatogram. Chromatograms of mixtures of propositus and normal fibrinogen confirmed the ability of this technique to distinguish normal from Baltimore fibrinogen. Chromatograms of fibrinogen isolated from two affected daughters displayed the characteristic increased anionic binding of peak 1 fibrinogen. Sedimentation velocity experiments indicated that the S(o) (20, [unk]) of fibrinogen Baltimore was slightly greater (8.13S vs. 7.85S) than that of normal fraction I-4. Differences in concentration dependence (- 0.65 c vs. - 1.30 c for normal) of the sedimentation coefficient could be attributable in part to spatial conformational differences. Molecular sieving experiments in acrylamide gels indicated that the molecular weight of propositus fraction I-2 was about the same as that of normal fibrinogen of comparable solubility (i.e. I-4, mol wt 325,000). Studies of the UV spectra, tyrosine/tryptophan ratios, sialic acid and hexose content, and N-terminal amino acids demonstrated no consistent significant differences from normal fraction I-4.

Amino Acids↗
PubMed1992

Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy.

We have compared gas chromatography and mass spectrometry (GC-MS) analysis with the Limulus amebocyte lysate (LAL) assay to quantify native meningococcal lipopolysaccharides (LPS) in five patient plasmas containing greater than 5 micrograms/liter by LAL. 3-Hydroxy lauric acid (3-OH-12:0) was used as a specific lipid A marker of neisserial LPS. The quantitative LAL results were confirmed by GC-MS (r = 0.98, P = 0.006). Seven patient plasmas were centrifuged at 103,000 g and the sedimentation behavior of native LPS compared with reference plasma proteins and with apo A1 and apo B100 representing high and low density lipoproteins. After 15 min of centrifugation, 84 +/- 2% (mean +/- SE) of the recovered LPS were found in the lower one-third of the centrifuged volume, whereas 6 +/- 1% remained in the upper one-third volume, indicating that meningococcal endotoxin circulates as complexes with high sedimentation coefficients. Bacterial outer membrane fragments were detected in the bottom fractions of three patient plasmas examined by means of electron microscopy. In three patient plasmas ultracentrifuged for 60 min at 103,000 g, the levels of apo A1 and apo B100 revealed minor changes, whereas only 1 +/- 1% of the recovered LPS remained in the upper one-third and 91 +/- 2% were found in the lower one-third volume. Few bioreactive LPS appear to be complexed with high and low density lipoproteins in meningococcal septic shock plasma.

Endotoxins↗
PubMed1998

A simple method for assessing copper-mediated oxidation of very-low-density lipoprotein isolated by rapid ultracentrifugation.

The association of very-low-density lipoprotein (VLDL) with atherosclerosis remains controversial. However, studies have shown that oxidative modification of VLDL can promote foam cell formation, leading to the development of atherosclerosis. A rapid method is described which will allow the significance of VLDL oxidation to be assessed in clinical studies. VLDL was isolated from heparinized plasma by a 1-h, single spin ultracentrifugation. Total protein was standardized to 25 mg/L. Oxidation was promoted by the addition of copper ions (17.5 mumol/L, final concentration) incubated at 37 degrees C. Conjugated diene production was followed at 234 nm. Total assay preparation time was 2 h. Urate greatly inhibited the oxidation of VLDL and was successfully removed by size exclusion chromatography. VLDL isolated from frozen plasma (-70 degrees C) was stable for 15 weeks. This simple, rapid method for the isolation of VLDL may be applied to assess the significance of VLDL oxidation in disease.

Copper↗
PubMed2005

Separation of normoblasts from whole blood by ultracentrifugation on arabino-galactan discontinuous gradients: A tool for prenatal diagnosis.

Normoblasts can be separated at high resolution from whole blood by ultracentrifugation on discontinuous gradients of arabino-galactane after the red blood cells are removed by sedimentation. With this method, isolation of fetal normoblasts from maternal blood could provide a tool for rapid prenatal diagnosis. The availability of this noninvasive technique could avoid the cost and the risks of miscarriage that are associated with the current invasive procedures.

Anemia, Sickle Cell↗
PubMed2006

Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals.

In developing and manufacturing protein biopharmaceuticals, aggregation is a parameter that needs careful monitoring to ensure the quality and consistency of the final biopharmaceutical drug product. The analytical method of choice used to perform this task is size-exclusion chromatography (SEC). However, it is becoming more and more apparent that considerable care is required in assessing the accuracy of SEC data. One old analytical tool that is now reappearing to help in this assessment is analytical ultracentrifugation (AUC). Developments in AUC hardware and, more importantly, recent developments in AUC data analysis computer programs have converged to provide this old biophysical tool with the ability to extract very high resolution size information about the molecules in a given sample from a simple sedimentation velocity experiment. In addition, AUC allows sample testing to be conducted in the exact or nearly exact liquid formulation or reconstituted liquid formulation of the biopharmaceutical in the vial, with minimal surface area contact with extraneous materials. As a result, AUC analysis can provide detailed information on the aggregation of a biopharmaceutical, while avoiding many of the major problems that can plague SEC, thus allowing AUC to be used as an orthogonal method to verify SEC aggregation information and the associating properties of biopharmaceuticals.

Biopharmaceutics↗
PubMed1982

Characterization of somatomedin binding in human serum by ultracentrifugation and gel filtration.

It is known that the somatomedins exist in human serum complexed to specific binding proteins. The existence of unbound somatomedins in serum has never unequivocally been demonstrated. We have characterized the distribution of insulin-like growth factor (IGF) I in different fractions after gel filtration of serum through Sephadex G-200 in neutral buffer. IGF-I was measured by RIA after acid extraction. Seventy-two percent of serum IGF-I was associated with large complexes with an estimated size of about 150,000 daltons and 25% was associated with smaller complexes of about 50,000 daltons. No unbound IGF-I was detected. Ultracentrifugation of 10 ml fresh serum was carried out at 106,000 X g for 17 h, after which the tube was aspirated in 1-ml fractions beginning at the top. IGF-I by RIA in fractions 2 and 3 sedimented with albumin; in fractions 4 to 7, the sedimentation pattern approached that of immunoglobulin G. This shift is consistent with the size distribution of IGF-I complexes demonstrated by gel filtration. The failure to find any significant increase in the concentration of IGF-I relative to albumin in the top 30% of the tube (fractions 1-3) after centrifugation argues against the presence of measurable free IGF-I in these fractions. The ability of upper fractions to bind added [125I]IGF-II proved to closely approximate the binding of the initial serum, indicating little sedimentation of the accessible binding protein. The relative binding of [125I]IGF-II by serum aliquots proved to be markedly concentration dependent. At concentrations above 5% serum, the incremental increase of binding as a function of serum concentration was much reduced. We interpret this to indicate that with dilution there is a dissociation of complexes and an increase in accessible binding sites. This phenomenon may modify tissue delivery of somatomedins in interstitial fluid. The data suggest that in undiluted serum there is no significant concentration of free somatomedins but at the dilution of serum that exists in the interstitial fluid, dissociation of bound somatomedins may be facilitated.

Adult↗
PubMed1999

[Applications of analytical ultracentrifuge to molecular biology and pharmaceutical science].

Analytical ultracentrifuges, XL-A and XL-I, developed by Beckman Company now find a broad application not only in universities but also in industries. Especially they are utilized conveniently in industries aiming at the development of proteins as a therapeutic drug or in those targeting drugs composed of small molecules developed on the basis of the structures of proteins. Sedimentation techniques can be used 1) to determine the molecular weight of proteins in solution, 2) to examine protein aggregation, 3) to evaluate the molecular shape of proteins, 4) to study the interaction of proteins, e.g. between ligands and receptors, and 5) to obtain insight into biological functions of homologous proteins. Application of this technique to molecular biology and pharmaceutical science will be reviewed with ample examples.

Agglutination↗
PubMed2005

Sedimentation velocity method in the analytical ultracentrifuge for the study of protein-protein interactions.

Sedimentation analysis in the analytical ultracentrifuge can be employed to detect macromolecular interactions. Whenever two molecules interact the mass of the resulting complex is increased and this is reflected in the sedimentation behavior. In this chapter we discuss how this phenomenon can be utilized to determine quantitative parameters of an interaction. An example, interaction of single-stranded DNA binding protein with a subunit of DNA polymerase III holoenzyme is given together with a thorough treatment of the relating theory and a description of evaluation algorithms.

Algorithms↗
PubMed1984

An ultracentrifuge study on the self-association of glucose dehydrogenase from Bacillus megaterium.

The self-association of glucose dehydrogenase (beta-D-glucose:NAD(P) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium was studied by analytical ultracentrifugation. The pH and composition of the buffer used were such that, owing to a reversible partial dissociation of the tetrameric enzyme, enzyme activity was reduced. It was found that under these conditions the protein exists in a monomer/dimer/tetramer association equilibrium.

Bacillus megaterium↗