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At least 721 records · Page 40Linked to original sources
PubMed1985

The separation of sheep and human serum "A"-esterase activity into the lipoprotein fraction by ultracentrifugation.

Using sheep and human serum the relationship between centrifugation time and yield of total lipoprotein, HDL-cholesterol and "A"-esterase in lipoprotein was studied employing different centrifuge rotors. More rapid separation of these components was obtained with a vertical rotor than with an angled rotor. The procedures commonly employed for lipoprotein separation gave low yields of lipoprotein "A"-esterase and HDL-cholesterol. The separation of sheep serum "A"-esterase into the lipoprotein fraction was not in phase with that of HDL-cholesterol and the pattern of separation was different from that in human serum. These results provide further evidence that serum "A"-esterase activity is associated with different species of HDL-particle.

Animals↗
PubMed1986

Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure.

A new two-step gradient technique has been used in the separation of the different classes of lipoproteins from the serum of cows, horses, dogs, pigs, rabbits and rats. Total lipoproteins were first isolated at d 1.21 then floated through a d 1.006 to d 1.21 gradient. Collection by mean of a gradient fractionator provided directly comparable lipoprotein profiles, allowed the determination of the exact density range of each lipoprotein class and the fraction by fraction analysis of composition. Cholesterol and apo AI recoveries were high. Horse, dog, rabbit and pig exhibited three distinct lipoprotein classes: VLDL, LDL and HDL. LDL were polydisperse in the pig (three components), light in the rabbit and scarce in the horse. In the Sprague-Dawley rat, LDL could not be individualized from HDL. In the bovine, LDL overlapped with a light form of HDL. Although AI was the main apoprotein in the HDL of all species, it ranged in proportion from 35% in the rat to 75% in the bovine. Apo AII was dimeric in the dog, as already known, but also in the horse, rabbit and bovine (MW:17,000) and in the pig MW:13,000). Apo AIV was present in the heavier HDL of all species. Rabbit, horse and pig HDL contained only one species of apo C which, in the pig was identified as apo CII.

Animals↗
PubMed2002

Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems.

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.

Animals↗
PubMed1985

The representation of equilibrium solute distributions for nonideal polydisperse systems in the analytical ultracentrifuge. Application to mucus glycoproteins.

It is relatively easy to represent by computer simulation the observed Rayleigh equilibrium fringe data for systems that are both associative and nonideal in the thermodynamic sense, and to extract the determinant parameters (see, for example, Roark, D., and D. A. Yphantis, 1969, Ann. NY Acad. Sci., 164:245-278; and Johnson M. L., J. J. Correia, D. A. Yphantis, and H. R. Halvorson, 1981, Biophys. J., 36:575-588). It is, however, considerably more difficult to represent systems that are both polydisperse (namely, those that consist of noninteracting species of different molecular weight) and nonideal, although the ideal case has been well described (see, for example, Tindall, S. H., and K. C. Aune, 1982, Anal. Biochem. 120:71-84). Here we show that the representation of nonideal polydisperse systems is now possible, after certain assumptions, by using a two-part interdependent minimization routine that uses readily available numerical packages. The method is applied to a well-characterized mucus glycoprotein (Mr approximately 2 X 10(6)) from the bronchial secretion of a cystic fibrosis patient. An excellent fit to the observed fringe data is obtained for a polydisperse three-component system, with a value for the second virial coefficient, B, of 0.57 ml mol g-2.

Biophysical Phenomena↗
PubMed2003

Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation.

The Escherichia coli UvrD protein (helicase II) is an SF1 superfamily helicase required for methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized quantitatively the self-assembly equilibria of the UvrD protein as a function of [NaCl], [glycerol], and temperature (5-35 degrees C; pH 8.3) using analytical sedimentation velocity and equilibrium techniques, and find that UvrD self-associates into dimeric and tetrameric species over a range of solution conditions (t</=25 degrees C). Increasing [NaCl] from 20mM to 200 mM decreases the dimerization equilibrium constant (L(20)) from 2.33(+/-0.30) microM(-1) to 0.297(+/-0.006) microM(-1) (pH 8.3, 20% (v/v) glycerol, 25 degrees C). The overall tetramerization equilibrium constant (L(40)) is 5.11(+/-0.80) microM(-3) at 20mM NaCl, but decreases so that it is not measurable at 200 mM NaCl. At 500 mM NaCl, only UvrD monomers are detectable. Increasing [glycerol] over the range from 20% to 40% (v/v) decreases both L(20) and L(40). We find no evidence for hexamer formation, although a species consistent in size with an octamer is detected at 35 degrees C. Inclusion of either ADP or ATPgammaS does not affect either L(20) or L(40) significantly, and does not induce the formation of additional assembly states. We also investigated the stoichiometry of UvrD binding to a 3'-(dT)(20)-18 bp DNA substrate by sedimentation equilibrium. At saturating concentrations of UvrD, three UvrD monomers can bind to the DNA substrate, although only two UvrD monomers are required to form a processive helicase complex. When the total DNA substrate concentration is about twofold greater than the total UvrD concentration, the vast majority of the DNA is bound by a single UvrD monomer.

Adenosine Triphosphatases↗