scholarly journals The use of equilibrium-density-gradient methods for the preparation and characterization of blood-group-specific glycoproteins

1970 ◽  
Vol 117 (5) ◽  
pp. 879-891 ◽  
Author(s):  
J. M. Creeth ◽  
M. A. Denborough
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Blood Group ◽  
Buoyant Density ◽  
Gradient Methods ◽  
Apparent Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Caesium Chloride ◽  
Selective Solvation

1. The method of sedimentation equilibrium in a gradient of caesium chloride has been applied to the preparation of blood-group-specific glycoproteins from human ovarian-cyst fluids: it is shown that virtually complete separation from contaminating protein is easily accomplished in a single step. 2. The glycoproteins isolated in this way have been characterized by analytical density-gradient experiments in both caesium chloride and caesium sulphate and values of the buoyant density, selective solvation and apparent molecular weight have been obtained. 3. In some cases, materials prepared from the same cysts by solvent extraction methods have also been characterized in these terms. 4. The selective solvation values are about 0.1 and 0.5g of water/g of glycoprotein in caesium chloride and caesium sulphate respectively. 5. The apparent molecular-weight values are much lower than the weight-average molecular weights, and it is shown that the origin of the discrepancy is heterogeneity in density of the glycoproteins. 6. Some sources of error in the interpretation of density-gradient schlieren patterns are examined.

scholarly journals The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation

1974 ◽  
Vol 143 (3) ◽  
pp. 669-679 ◽  
Author(s):  
K. Ramakrishnan Bhaskar ◽  
J. Michael Creeth
Keyword(s):  
Density Gradient ◽  
Blood Group ◽  
Buoyant Density ◽  
Physicochemical Characterization ◽  
Cyst Fluid ◽  
Equilibrium Density ◽  
Density Gradient Ultracentrifugation ◽  
Molecular Weights ◽  
Molar Ratios ◽  
Blood Group Substances

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete ρ0 values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Lea specificity. All fractions had approximately equal Lea activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8–0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.

scholarly journals The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation

1974 ◽  
Vol 143 (1) ◽  
pp. 159-170 ◽  
Author(s):  
J. Michael Creeth ◽  
K. Ramakrishnan Bhaskar ◽  
Alastair S. R. Donald ◽  
Walter T. J. Morgan
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Blood Group ◽  
Buoyant Density ◽  
Extraction Procedure ◽  
Sedimentation Velocity ◽  
Water Soluble ◽  
Solvent Fractionation ◽  
Cross Linkage ◽  
Serological Activity

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH4)2SO4 and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH4)2SO4.

scholarly journals Some Physicochemical Properties of Erysimum Latent Virus

1982 ◽  
Vol 35 (1) ◽  
pp. 5
Author(s):  
Keith H Gough ◽  
Glenn G Lilley ◽  
Dharma D Shukla ◽  
Frank Woods
Keyword(s):  
Molecular Weight ◽  
Buoyant Density ◽  
Latent Virus ◽  
Sedimentation Equilibrium ◽  
Protein Subunit ◽  
Molecular Weights ◽  
Caesium Chloride ◽  
Rna Content ◽  
Velocity Diffusion ◽  
Phosphorus Determination

Sedimentation velocity, diffusion coefficient and sedimentation equilibrium measurements gave a molecular weight of 5 �90 x 106 for the intact Erysimum latent virus. The molecular weight of the empty shell was estimated to be 3�92 X 106 and the protein subunit to be 21 600. The RNA content calculated from the molecular weights of the full and empty particles is 33 %, in agreement with that estimated from the buoyant density in caesium chloride. However, a direct phosphorus determination gave an RNA content of only 28 %.

scholarly journals Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 199 (3) ◽  
pp. 581-589 ◽  
Author(s):  
John K. Sheehan ◽  
Ingemar Carlstedt ◽  
Lars Cöster ◽  
Anders Malmström ◽  
Lars-Ȧke Fransson
Keyword(s):  
Molecular Weight ◽  
Buoyant Density ◽  
Broad Band ◽  
Apparent Molecular Weight ◽  
Gel Chromatography ◽  
Analytical Ultracentrifuge ◽  
Dermatan Sulphate ◽  
Caesium Chloride ◽  
Mode 2 ◽  
Isopycnic Centrifugation

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs2SO4 both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density ρ=1.75g/ml whereas fraction PG-II banded sharply at ρ=1.64g/ml. However, in Cs2SO4, fraction PG-II banded at ρ=1.51g/ml and fraction PG-I at ρ=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs2SO4 fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs2SO4 gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88–42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs2SO4 were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1×106) than were those obtained previously for their ‘monomeric’ states (fraction PG-I 160000–410000, and fraction PG-II 70000–130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs2SO4 (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs2SO4. Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

scholarly journals Macromolecular distribution near the limits of density-gradient columns. Some applications to the separation and fractionation of glycoproteins

1977 ◽  
Vol 161 (3) ◽  
pp. 449-463 ◽  
Author(s):  
M J Creeth ◽  
J R Horton
Keyword(s):  
Molecular Weight ◽  
Density Distribution ◽  
Density Gradient ◽  
Specific Volume ◽  
Buoyant Density ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Macromolecular Component

1. Expressions are derived for the distribution at density-gradient equilibrium of macromolecules whose densities are (a) close to the values characterizing the solution limits or (b) outside the span of the gradient. 2. Density-distribution predicted by the expressions agree with those obtained by rigorous methods. 3. The distribution equations are applied to hypothetical mixtures of proteins and glycoproteins in commonly used density-gradient media to simulate separation and fractionation conditions. 4. It is shown that CsBr, although less efficient than CsCl for fractionation, is nevertheless adequate for most purposes; in analytical experiments it may often have advantages over CsCl. Limitations on the use of LiBr are explored. 5. An expression is derived which allows the variance of the partial specific volume of the macromolecular component to be determined from the variance of the buoyant density. It is shown that the relative resolving powers of different salts is expressed by their values of the quantity (formula: see text). 6. The equations are applied to a well-characterized glycoprotein preparation at equilibrium in CsCl and in Cs2SO4:it is shown that the much wider distribution in CsCl than in Cs2SO4 is explicable in terms of the variance in buoyant density and the solvation properties of the salts. 7. Limitations of the expressions arise when dispersity in density is represented by a low apparent molecular weight; realistic simulations can then only be obtained when the component is fully banded.

scholarly journals Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila

1983 ◽  
Vol 209 (2) ◽  
pp. 461-470 ◽  
Author(s):  
P Londei ◽  
A Teichner ◽  
P Cammarano ◽  
M De Rosa ◽  
A Gambacorta
Keyword(s):  
Molecular Weight ◽  
Average Molecular Weight ◽  
Buoyant Density ◽  
Protein Composition ◽  
Equilibrium Density ◽  
Number Average Molecular Weight ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Protein Components ◽  
Protein Number

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6×10(6) Da, compared with 20 proteins totalling 0.35×10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15×10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87×10(6) Da mass); a smaller difference. 0.15×10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8×10(6) and 1.65×10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.

scholarly journals Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants

1969 ◽  
Vol 112 (5) ◽  
pp. 777-786 ◽  
Author(s):  
Richard Wells ◽  
Max Birnstiel
Keyword(s):  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Gaussian Distribution ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Higher Plants ◽  
Buoyant Density ◽  
Cell Fractionation ◽  
Caesium Chloride ◽  
Cellular Dna

1. Chloroplasts and mitochondria were isolated by aqueous and non-aqueous cell-fractionation techniques. In a variety of higher plants the mitochondrial DNA bands in a caesium chloride gradient at 1·706g.cm.−3, whereas chloroplastal DNA has a buoyant density of 1·697g.cm.−3. 2. In total cellular DNA of moderate molecular weight, the chloroplastal DNA is found within the Gaussian distribution of the nuclear DNA and is not resolved as a satellite. 3. Both chloroplastal DNA and mitochondrial DNA from lettuce renature rapidly. 4. The kinetic complexity of mitochondrial DNA is > 108 daltons. 5. Chloroplastal DNA is made up from fast and slow renaturing sequences with kinetic complexities of 3×106 and 1·2×108 daltons respectively. 6. From the discrepancy between analytical and kinetic complexity it is concluded that chloroplastal DNA is extensively reiterated.

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