Chromatographic depletion of lipoproteins from plasma and recovery of apolipoproteins

Carson, Steven D.

doi:10.1016/0005-2760(83)90034-6

Cited by 10 publications

(3 citation statements)

References 15 publications

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“…Adipocytes (10,000 cells) were seeded in a 24-well plate in DMEM with 10% calf serum overnight. Cells were serum-deprived for 4 h, treated with 100 nM insulin for 3.5 h, and then with 50 μM of [ 14 C]oleate in HBS containing 0.1% fatty acid-free BSA for 30 min 51,52 . Cells were washed extensively in cold HBS with 0.1% fatty acid-free BSA to remove unincorporated [ 14 C]oleate, lysed in RIPA buffer (Thermo Fisher), and centrifuged at 2000 rpm for 5 min.…”

Section: Methodsmentioning

confidence: 99%

EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice

Arif

Terenzi

Potdar

et al. 2017

Nature

111

122

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Metabolic pathways contributing to adiposity and aging are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis1–3. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting additional downstream effectors4–6. Here we identify glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes importantly to adiposity and aging. EPRS phosphorylation at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex (MSC), required for execution of noncanonical functions beyond protein synthesis7,8. To investigate physiological function of EPRS phosphorylation, we generated EPRS knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, thereby displaying notable similarities with S6K1-deficient mice9–11 and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent12. Substitution of the EPRS S999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the MSC. Interaction screening revealed phospho-EPRS binds Slc27a1 (i.e., fatty acid transport protein 1, FATP1)13–15, inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors critical for metabolic phenotypes.

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Section: Methodsmentioning

confidence: 99%

EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice

Arif

Terenzi

Potdar

et al. 2017

Nature

111

122

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show abstract

“…The salt concentration of the lipoprotein fraction was reduced to .5 M NaCl by a 1:5 dilution with TRIS [tris(hydroxymethyl)aminomethanej-saline (.05 M TRIS, .1 M NaCl, .01% NaN 3 , pH = 7.6). The resulting 20-mL sample was pumped onto a phenyl-Sepharose (Pharmacia, Piscataway, NJ) column (1.5 x 26 cm) equilibrated with TRIS-saline for hydrophobic interaction chromatography (Carson, 1983;Ross and Carson, 1985). The column was washed with TRIS-saline at 20 mL/h to remove die nonadsorbed plasma protein fraction.…”

Section: Methodsmentioning

confidence: 99%

“…The secondary structure of the delipidated protein was more sensitive to added lipid, exhibiting an increase in helix from 48% in the absence of lipid to 58% when the phospholipid-to-protein ratio was 6 to 1. Carson (1983) used hydrophobic interaction chromatography to prepare lipoprotein-free plasma, the principle being that lipoproteins were adsorbed via the lipid moiety from whole plasma onto the hydrophobic resin. Plasma components other than lipoproteins were removed from the resin with a buffered saline wash. Apolipoproteins were then eluted from the phenyl-Sepharose with a propylene glycol gradient, whereas the lipid components remained bound to the resin.…”

Section: Table 1 Amino Acid (Aa) Composition Of Chicken High Densitymentioning

confidence: 99%

Effects of Phospholipids on the Conformation of Chicken High Density Lipoprotein

Brown

1989

Poultry Science

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Apolipoprotein A-I (apo-A-I), the major protein component of chicken high density lipoprotein (HDL), was isolated by hydrophobic interaction chromatography from the lipoprotein fraction of plasma. The apo-A-I was identified from its amino acid composition and molecular weight (by electrophoresis). The isolated protein contained 15% lipid, in the form of neutral lipids and free fatty acids, as compared with 50% lipid, including phospholipid, in intact HDL. The conformation of the isolated protein, as determined from the circular dichroism spectrum, was essentially that of the protein in intact chicken HDL, and similar to that of human HDL. The addition of phospholipid had little effect on the spectrum of this protein. Total delipidation of the protein by extraction with ethanol-diethyl ether mixtures removed the residual lipid, thereby producing an apoprotein with decreased helical structure. The secondary structure of this apoprotein could be restored by the addition of phospholipid.

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Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma

Ross¹,

Carson²

1985

Analytical Biochemistry

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Chromatographic depletion of lipoproteins from plasma and recovery of apolipoproteins

Cited by 10 publications

References 15 publications

EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice

EPRS is a critical mTORC1–S6K1 effector that influences adiposity in mice

Effects of Phospholipids on the Conformation of Chicken High Density Lipoprotein

Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma

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