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At least 415 records · Page 23Linked to original sources

PubMed1981

Accuracy and reproducibility of a quantitative electrophoresis of plasma lipoproteins: interlaboratory comparison and verification by ultracentrifugation.

Fifty-three samples of sera from normal subjects and from patients with type IIa, IIb and IV hyperlipoproteinemia were shipped from Hannover to the collaborative laboratories in Vienna and Mannheim, which participated in the quantification of their lipoprotein fractions based on polyanion precipitation of electrophoretically separated lipoproteins followed by densitometric measurement of alpha-, prebeta- and beta-lipoproteins. Total serum cholesterol and cholesterol calculated from quantified lipoprotein fractions were highly correlated, the correlation coefficients ranged between r = 0.93-0.98. Furthermore, we found a high concordance comparing the beta-, and prebeta- and alpha-cholesterol with data obtained from lipoprotein fractionation and subsequent cholesterol measurement by ultracentrifugation. The obtained correlation coefficients were always higher than r = 0.91. High accuracy and reproducibility of the quantitative lipoprotein electrophoresis justify its recommendation for wide use in the field of clinical chemistry.

Cholesterol↗

PubMed1985

Fast separation of the three main plasma lipoprotein classes by ultracentrifugation using vertical rotor and multiple discontinuous density gradient.

An ultracentrifugation technique for isolation of the various lipoprotein fractions using a vertical rotor is described. By the use of a multiple discontinuous density gradient, very low density lipoproteins, low density lipoproteins and high density lipoproteins are sharply separated, without contamination of other lipoproteins or albumin. Centrifugation time is 80 min. The densities, electrophoretic mobilities, electron microscopic appearance and chemical composition are those of the expected classes of lipoproteins. Two different gradients are used to enhance the separation of very low density lipoproteins from low density lipoproteins on one hand and high density lipoproteins from infranatant on the other.

Centrifugation, Density Gradient↗

PubMed1990

Rapid isolation of low density lipoprotein (LDL) subfractions from plasma by density gradient ultracentrifugation.

High resolution density gradient ultracentrifugation (DGUC) and non-denaturing gradient gel electrophoresis (GGE) indicate that low density lipoprotein (LDL) in both normal and hyperlipidaemic subjects is composed of overlapping particle populations. A new centrifugation procedure has been developed which permits the separation of LDL subspecies directly from plasma within 24 h. The profiles obtained were analogous to those seen on gradient gel electrophoresis. LDL was divided into 3 fractions. The plasma concentration of LDL-I seen in young females was twice that in men (85.6 +/- 28.8 vs. 42.3 +/- 25.7 mg/dl, P less than 0.005). LDL-II was not significantly different in any group while LDL-III was specifically elevated in coronary artery disease (CAD) patients (207.1 +/- 92.6 mg/dl in CAD vs. 87.4 +/- 79.6 mg/dl in normal men, P less than 0.05). The presence of small, dense LDL detected either by density gradient centrifugation or gel electrophoresis was associated with raised triglyceride (TG) and low high density lipoprotein (HDL) cholesterol and may be a risk marker for coronary artery disease.

Adolescent↗

PubMed1990

Determination of size and molecular weight distributions of lipoproteins using automatic image analysis and density gradient ultracentrifugation.

A Leitz-Tas automatic image analysis system has been used to investigate the size distributions of human lipoprotein particles isolated by density gradient ultracentrifugation. Computerized image analysis enabled us to obtain more precise determinations of modal diameter and overall range in diameter for each lipoprotein subspecies than those determined by classical methods. In addition, lipoprotein molecular weight distributions were calculated from measurements of flotation density and particle size distributions. This method offers certain advantages over other procedures used to characterize mean molecular weight with regard to the ability to define lipoprotein polydispersity. The method and its limitations are discussed and illustrated with results obtained with human lipoproteins.

Adult↗

PubMed1983

Partial characterization of circulating macromolecular beta 2-microglobulin complexes by density gradient ultracentrifugation and page-blotting.

beta 2-microglobulin (beta 2m)-containing, rapidly sedimenting peaks were seen when sera of patients with 3% polyethylene glycol (PEG)-insoluble beta 2m were fractionated by sucrose density gradient ultracentrifugation. No such material was found in serum of a healthy volunteer without 3% PEG-insoluble beta 2m. MW of beta 2m-containing high molecular weight fractions ranged from 1.6 X 10(5) to 2.1 X 10(6). When some fractions were recentrifuged a majority of beta 2m was recovered at the original sedimentation position. Analysis of high molecular weight beta 2m-containing peaks by SDS-PAGE followed by electroblotting revealed only monomeric beta 2m indicating that beta 2m was not covalently bound in the high molecular weight material.

Antigen-Antibody Complex↗

PubMed1990

Purification of poliovirus 14 S subunits by sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography.

Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.

Antibodies, Monoclonal↗

PubMed1975

Determination of the equilibrium constants of self-associating protein systems. XI. The application of C-(r) in graphical analysis and the enumeration of interacting species in the ultracentrifuge.

A greatly simplified procedure is proposed which employs C equals f(r) as determined from sedimentation equilibrium measurements in graphical analysis of self-associating protein systems and in the enumeration of interacting species in the ultracentrifuge. Basic equations given here are applicable to any self-associating system. A procedure is outlined for enumeration of interacting components independent of non-ideal behavior, using principal component analysis.

Binding Sites↗

PubMed1984

Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. I. Experimental testing of previous models.

The 'microheterogeneity model' (R.J. Siezen and R. van Driel, Biochim. Biophys. Acta 295 (1973) 131) and the 'incompetent whole molecule model' (G. Kegeles, Arch. Biochem. Biophys. 180 (1977) 530) for the dissociation of Helix pomatia alpha-hemocyanin whole molecules to half molecules were tested experimentally, using ultracentrifugation and stopped-flow dilution analysis. Results of differential sedimentation experiments followed by stopped flow analysis of separated fractions of 60 S and 100 S molecules were not entirely as predicted by the incompetent model, the agreement depending on the pH and ionic strength of analysis. A considerable amount of stopped-flow dilution response could be attributed to material sedimenting between 60 and 100 S. This material appears to be the main equilibrating fraction, and its amount is considerably larger than that predicted by the microheterogeneity model. Increased hydrostatic pressure was found to enhance this fraction, whereas fixation or low ionic strength reduced or eliminated this fraction. Nonequilibrium components of 30, 50 and 80 S were detected and partially purified by differential sedimentation.

Journal Article↗

PubMed1984

Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. II. Simulations of reacting microheterogeneous hemocyanin.

Simulations for the moving-boundary ultracentrifuge behavior of Helix pomatia alpha-hemocyanin have been developed, based on modification of published models. In the present treatment, it has been assumed that the protein system is extensively microheterogeneous, with respect to its whole-half molecule reactivity. It has also been assumed that all such association and dissociation reactions can proceed to equilibrium in a time appreciably shorter than that which would be required to separate nonreacting half molecules from nonreacting whole molecules. Predictions based on this model agree well with reported experimental findings of nonequilibration of fractionated material, apparent independence of whole-to-half molecule concentration ratios on total concentration in sedimentation experiments, and stopped-flow dilution reactivity over a wide range of sedimentation coefficients.

Journal Article↗

PubMed1987

Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation.

The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NACl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively.

Journal Article↗

PubMed1976

Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients, an improved, simplified method.

A mathematical method is described for calculating the sedimentation coefficient (S20,W) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evaluation (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.

Centrifugation, Density Gradient↗

PubMed2006

An ultracentrifugation-based approach to the detection of hepatitis A virus in soft fruits.

A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.

Centrifugation↗

PubMed2006

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation.

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.

Blotting, Western↗

PubMed2003

Molecular fractionation of starch by density-gradient ultracentrifugation.

Amylose and amylopectin in corn and potato starches were fractionated by centrifugation at 124,000g for 3-72 h at 40 degrees C in a gradient media, Nycodenz, based on their sedimentation rate differences. The fractions were collected from a centrifuge tube, and then analyzed by the phenol-sulfuric acid method and iodine-binding test. Amylopectin, a large and highly branched starch molecule, migrated faster than amylose and quickly reached its isopycnic point with a buoyant density of about 1.25 g/mL, exhibiting a sharp and stable carbohydrate peak. Amylose, which is a relatively small and linear molecule, however, migrated slowly in a broad density range and continued moving to higher density regions, eventually overlapping with amylopectin peak as the centrifugation continued. This could indicate that the buoyant density of amylose is similar to that of amylopectin. Under centrifugal conditions of 3 h and 124,000g, amylose and amylopectin molecules were clearly separated, and the presence of intermediate starch molecules (11.5 and 7.7% for corn and potato starch, respectively) was also observed between amylose and amylopectin fractions. The amylose content of corn and potato starches was 22.6 and 21.1%, respectively, based on the total carbohydrate analysis after the ultracentrifugation for 3 h. In alkaline gradients (pH 11 or 12.5), the sedimentation rate of starch molecules and the buoyant density of amylopectin were reduced, possibly due to the structural changes induced by alkali.

Amylopectin↗

PubMed1985

Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium.

A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing.

Animals↗

PubMed1997

Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation.

BACKGROUND/AIMS: Cholesterol crystallization in a dilute, bile salt-rich model bile is a multiphase process in which early filamentous crystals gradually transform to classical cholesterol monohydrate plates. The pertinence of similar transformations in more complex model systems or native bile is, however, unclear. The aim of the present study was to characterize and monitor cholesterol crystallization in a model bile of physiological relevance. METHODS: A supersaturated model bile was prepared with a lipid composition (18 mM cholesterol, 37 mM lecithin, 120 mM taurocholate) that was derived from analyzing 10 gallbladder biles from cholesterol gallstone patients. Cholesterol crystallization was followed by light and electron microscopy, and sequential density gradient analysis of cholesterol-containing precipitates. RESULTS: During cholesterol crystallization a reproducible sequence of events was recorded. First (T<18 h), cholesterol-rich vesicular and multilamellar structures (density 1.005-1.015 g/ml) were observed. Later, (T>60 h) filamentous, helical, tubular (density 1.015-1.04 g/ml) and plate-like (density 1.04-1.06 g/ml) cholesterol crystals appeared. The concentration of crystals increased gradually, while bilayer structures became desaturated with cholesterol and disappeared, and early crystal forms were replaced by plates. Eventually (T>25 days) only classical plate-like cholesterol monohydrate crystals were present. Exposure of cholesterol-containing precipitates to micellar (100 mM) deoxycholate dissolved the bilayer structures but not the crystals. CONCLUSIONS: These data demonstrate that cholesterol crystallization in a physiologically relevant model bile is a multiphase process consisting of a sequence of transitions from vesicular and multilamellar structures to early crystal forms and to classical plate-like cholesterol monohydrate crystals. These transitions are associated with increasing density and decreasing phospholipid content of cholesterol precipitates. We suggest that time-lapse density gradient ultracentrifugation is a useful method for investigating and quantitating the process of cholesterol crystallization and factors that influence this process in bile.

Animals↗

PubMed2002

An ultracentrifugation analysis of two hundred fish genomes.

The goal of this study was to provide a comprehensive view of the compositional characteristics of fish genomes. We therefore expanded the number of fish species that we had explored so far in their DNAs by analytical ultracentrifugation in CsCl density gradient from 122 to 201. This study included representatives from three out of nine orders of Elasmobranchs (sharks and rays), both orders of dipnoan lungfishes, and both orders of chondrosteans (sturgeons and bichirs). We also studied 19 out of 38 teleostean orders, which represent all but four (minor) superorders of the subdivision Teleostei, a group comprising about 23,600 species (96% of all extant fishes). This leaves for further studies two subclasses, Holocephali (chimaeras), and Coelacanthimorpha (gombessas). In spite of this substantial increase in the number of species and orders analysed, all average properties (the modal buoyant density, rho(0), the average buoyant density, , the CsCl profile asymmetry, A, and the compositional heterogeneity, H), and all their ranges were unchanged compared to a previous study [J. Mol. Evol. 31 (1990) 265]. This suggests that, in all likelihood, the properties reported in the present paper can be considered as generally valid for all fish genomes.

Animals↗

PubMed2005

Metal ion complexes of EDTA as solutes for density gradient ultracentrifugation: influence of metal ions.

In the study reported here, we study the nature of the metal ion complexes of EDTA as solute systems for analysis of lipoproteins by density gradient ultracentrifugation (DGU) by varying both the complexing metal ion and the counterion. Specifically, the sodium and cesium salts of complexes of Bi/EDTA, Pb/EDTA, Cd/EDTA, Fe/EDTA, and Cu/EDTA were chosen for this study. We show that useful gradients can be formed within a few hours beginning with a homogeneous solution. Data are presented that provide insight into the nature of how these gradients are formed from these complexes and how the selection of a specific complex can be used to enhance particular regions of the lipoprotein density profile for clinical studies. We also examine the use of equilibrium sedimentation theory to correlate the measured density profiles generated by these complexes with their molecular weight.

Centrifugation, Density Gradient↗