Analytical Equilibrium Density Gradient Ultracentrifugation of Human Plasma Lipoproteins

Nature ◽  
1964 ◽  
Vol 202 (4931) ◽  
pp. 490-491 ◽  
Author(s):  
GEORGE H. ADAMS ◽  
VERNE N. SCHUMAKER
Keyword(s):  
Human Plasma ◽  
Density Gradient ◽  
Plasma Lipoproteins ◽  
Equilibrium Density ◽  
Density Gradient Ultracentrifugation

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 Lipoprotein lipase is active as a monomer

2019 ◽  
Vol 116 (13) ◽  
pp. 6319-6328 ◽  
Author(s):  
Anne P. Beigneux ◽  
Christopher M. Allan ◽  
Norma P. Sandoval ◽  
Geoffrey W. Cho ◽  
Patrick J. Heizer ◽  
...  
Keyword(s):  
Density Gradient ◽  
Lipoprotein Lipase ◽  
High Salt ◽  
Plasma Lipoproteins ◽  
Density Gradient Ultracentrifugation ◽  
Sepharose Column ◽  
Low Salt ◽  
Catalytically Active ◽  
Lpl Mass

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Incorporation of radioactive phospholipid into subclasses of high-density lipoproteins

1983 ◽  
Vol 244 (5) ◽  
pp. E513-E516 ◽  
Author(s):  
A. R. Tall ◽  
C. B. Blum ◽  
S. M. Grundy
Keyword(s):  
Density Gradient ◽  
Specific Activity ◽  
High Density ◽  
High Density Lipoproteins ◽  
Equilibrium Density ◽  
Density Gradient Ultracentrifugation ◽  
Hdl Subfractions ◽  
Time Points ◽  
Plasma High Density

The incorporation of orally administered phospholipid into plasma high-density lipoproteins (HDL) was studied in three subjects. Plasma was analyzed by equilibrium density gradient ultracentrifugation, 5, 6, and 8 h after ingestion of 1.1 g [3H-choline, 14C-dilinoleoyl]phosphatidylcholine. At all time points in all subjects, there was a peak of phosphatidylcholine specific activity in fractions of density approximately 1.10-1.13 g/ml, corresponding to the subclass previously designated HDL2a. There was also a more variable, smaller peak of specific activity of phospholipids in HDL2b (1.063-1.100 g/ml) and in fractions of density approximately 1.19 g/ml. In the 1.10-1.13 fraction, 97 and 71%, respectively, of the 3H and 14C radioactivity were in phospholipids. The 3H/14C ratio was similar in phospholipids of HDL subfractions, the d less than 1.07 fraction, and in the administered phospholipid. The results show preferential transfer or exchange or absorbed phosphatidylcholine into specific subclasses of HDL.

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