Isolation of plasma lipoproteins by a combination of differential and density gradient ultracentrifugation

1988 ◽  
Vol 330 (2) ◽  
pp. 149-151 ◽  
Author(s):  
A. H. M. Terpstra ◽  
A. E. Pels
Keyword(s):  
Density Gradient ◽  
Plasma Lipoproteins ◽  
Density Gradient Ultracentrifugation

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.

scholarly journals Isolation of human synovial-fluid hyaluronate by density-gradient ultracentrifugation and evaluation of its protein content

1972 ◽  
Vol 126 (5) ◽  
pp. 1073-1080 ◽  
Author(s):  
Irwin Scher ◽  
David Hamerman
Keyword(s):  
Synovial Fluid ◽  
Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Proteolytic Digestion ◽  
Peptide Fragments ◽  
Guanidinium Chloride ◽  
Caesium Chloride ◽  
Normal Human ◽  
Protein Moiety ◽  
Human Synovial Fluid

1. A compound of hyaluronate and protein, called hyaluronate–protein was isolated from pooled human synovial fluids by caesium chloride density-gradient ultracentrifugation. 2. The isolated hyaluronate–protein was labelled with [125I]iodide and the following studies were done. (a) Ultracentrifugation in caesium chloride showed that the protein moiety (125I counts) and hyaluronate (hexuronate) sedimented together in the middle of the gradient. (b) The labelled hyaluronate–protein was treated with trypsin, and ultracentrifugation showed that peptide fragments (125I counts) were dispersed throughout the gradient, indicating proteolytic digestion. Hyaluronate sedimented in the middle of the gradient. (c) The labelled hyaluronate–protein was digested with streptococcal hyaluronidase, and ultracentrifugation showed that hyaluronate fragments were dispersed throughout the gradient, indicating digestion of the polysaccharide. The protein moiety, without attached hyaluronate, now sedimented at the top of the gradient. (d) Ultracentrifugation of labelled hyaluronate–protein in 4m-guanidinium chloride showed that protein and hyaluronate sedimented together. 3. These studies confirm that hyaluronate is combined with a small quantity of protein in normal human synovial fluid. A mild method for the rapid isolation of hyaluronate–protein in good yield is described.

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.

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