Sulfhydryl chemistry and solubility properties of human plasma apolipoprotein B

Cardin, Alan D.; Witt, Kathleen R.; Barnhart, Carole L.; Jackson, Richard L.

doi:10.1021/bi00261a048

Cited by 105 publications

(31 citation statements)

References 48 publications

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“…VLDL (density (d) < 1.006 g= ml) and HDL (1.070 < d < 1.21) were isolated from plasma by Apolipoproteins from each lipoprotein fraction were isolated by preparative discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis on a 3 and 15% gel. 12,13 Apolipoprotein bands were excised from polyacrylamide gels and hydrolysed in 6 M HCl at 110 C for 16 h under nitrogen. Samples were then centrifuged to remove polyacrylamide.…”

Section: Analytical Proceduresmentioning

confidence: 99%

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

et al. 2002

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AIMS=HYPOTHESIS:Mechanisms responsible for the decreased high-density lipoprotein (HDL) cholesterol level associated with insulin resistance in obese patients are not clearly understood. To determine the influence of insulin resistance at an early stage on HDL metabolism, we performed a stable isotope kinetic study of apolipoprotein (apo) A-I, in five obese insulin resistant women with normal fasting triglycerides and without impaired glucose tolerance, and in five age-matched control women. METHODS: Each subject received a 16 h constant infusion of L-[1-13 C]leucine at 0.7 mg=kg=h following a primed bolus of 0.7 mg=kg. RESULTS: ApoA-I fractional catabolic rate (FCR) was significantly increased in insulin-resistant women compared to controls (0.316 AE 0.056 vs 0.210 AE 0.040 per day, P < 0.01), indicating a significant 50% increase of apoA-I catabolism, leading to an important reduction of plasma apoA-I residence time (3.25 AE 0.59 vs 4.92 AE 1.11, P < 0.01). ApoA-I production rate tended to be higher in insulin resistant women than in controls (364 AE 77 vs 258 AE 60 mg=l=day, P ¼ 0.13), but the difference was not statistically significant. ApoA-I FCR was correlated with triglycerides during the fed state (r ¼ 0.69; P ¼ 0.026) and HDL triglycerides -esterified cholesterol ratio (r ¼ 0.73; P ¼ 0.016), suggesting that alteration of apoA-I metabolism in insulin resistance may be partly related to HDL enrichment in triglycerides. CONCLUSIONS: Our kinetic study shows that patients, at an early stage of insulin resistance (without impaired glucose tolerance nor fasting hypertriglyceridaemia), already have a significant alteration of apoA-I metabolism (increased apoA-I catabolism), which is consistent with the increased risk of atherosclerosis in this population.

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Section: Analytical Proceduresmentioning

confidence: 99%

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

et al. 2002

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“…Others have reported apparent Mrs of 250,000-550,000, based on sedimentation-equilibrium and gel-permeation properties in the presence of 6-7 M guanidine (1, 10, 11), NaDodSO4/PAGE (12), and stoichiometric titration of antigenic determinants on LDL particles (13). Progress in determining the amino acid sequence of apo B has been slow due to aggregation and insolubility of the delipidated protein in aqueous buffers (14,15). Recently, Staphylococcus aureus protease was used to cleave large peptides of human apo B-100 from LDL.…”

mentioning

confidence: 99%

“…Progress in determining the amino acid sequence of apo B has been slow due to aggregation and insolubility of the delipidated protein in aqueous buffers (14,15). Recently, Staphylococcus aureus protease was used to cleave large peptides of human apo B-100 from LDL.…”

mentioning

confidence: 99%

A partial cDNA clone for human apolipoprotein B.

Deeb¹,

Motulsky²,

Albers³

1985

Proc. Natl. Acad. Sci. U.S.A.

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A human liver cDNA library was screened for sequences coding for apolipoprotein B (apo B), the major protein of human low density lipoproteins. A mixture of synthetic oligonucleotides (26 bases long) coding for an amino acid sequence known to exist in apo B was used as a hybridization probe. A clone was identified that had a cDNA insert of 593 base pairs and that contained sequences coding for a peptide of 24 residues that had earlier been isolated from apo B by limited proteolysis. There is considerable variation in estimates of the molecular weight of the monomeric unit of apo B. Results from several laboratories suggest that apo B-100 is a multimeric protein with subunits ranging in apparent Mr between 20,000 and 80,000 (1, 7-10). Others have reported apparent Mrs of 250,000-550,000, based on sedimentation-equilibrium and gel-permeation properties in the presence of 6-7 M guanidine (1, 10, 11), NaDodSO4/PAGE (12), and stoichiometric titration of antigenic determinants on LDL particles (13). Progress in determining the amino acid sequence of apo B has been slow due to aggregation and insolubility of the delipidated protein in aqueous buffers (14,15). Recently, Staphylococcus aureus protease was used to cleave large peptides of human apo B-100 from LDL. Two of these peptides (24 and 25 amino acid residues long) were purified and sequenced (16). We have used a part of the amino acid sequence of one of these peptides to construct an oligonucleotide which we used to probe a human liver cDNA library for apo B sequences. Here we describe the isolation and characterization of recombinant plasmids containing cDNAs coding for a portion of apo B. The human liver cDNA library was kindly provided by K The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODS

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“…The purified fractions were sterile filtered over 0. 22 Millex filter (Millipore Co.) and stored at 4 °C. lodination of LDL LDL was 125 I-labeDed by the IC1 method of Karlin et al (25), purified over Sephadex G75 columns (20 l cm) and dialysed against Standard buffer.…”

Section: Chemical Analysismentioning

confidence: 99%

“…It was intended to prepare stable nucleoside-LDL conjugates and to modify these conjugates in order to utilize scavenger receptor transport. Electrophoresis SDS-PAGE was performed according to the method of Cardin et al (22) modified äs previously described (23). Agarose lipoprotein electrophoresis was conducted with the Paragon Lipo Kit (Fa.…”

Section: Introductionmentioning

confidence: 99%

Preparation of Nucleoside-LDL-Conjugates for the Study of Cell-Selective Internalization: Stability Characteristics and Receptor Affinity

Schultis

Baeyer²,

Neitzel³

et al. 1991

Clinical Chemistry and Laboratory Medicine

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Summary: Antiviral therapy of human immundeficiency virus (HIV) infection is currently based on Inhibition of reverse transcriptase by dideoxynucleosides, such äs azidothymidine. Because of widespread toxicity it is reasonable to selectively target these drugs to infected cells. This may be accomplished utilizing drug-LDL conjugates, which are interüalized via cell specific receptor pathways. With respect to HIV infection, scavenger receptors of the macrophage System seems to offer a hopefull perspective. This pathway requires chemical modification of surface polarity of the LDL. Cell experiments were conducted in HepG2 hepatocytes, which express apolipoprotein B receptors, and in P 388 macrophages, which express scavenger receptors. LDL particles to be conjugated were isolated from blood donor plasma and from LDL-apheresis waste material. Noncovalent LDL conjugation with amphiphilic nucleoside derivatives produced only an unspecific nucleoside transfer to cell membranes, due to instability of the LDL conjugates. An experimental method (coincubation test) was developed to identify those conjugates that are stable in the presence of other lipophilic compartments. Covalent coupling pf nuclepsides to the apolipoprotein B moiety of LDL particles resulted in stable conjugates. As a consequence, the surfaee Charge became negative, and the LDL displayed scavenger receptor affmity rather than apolipoprotein B receptor affinity. Selective targeting of nucleosides to macrophages can be accomplished by covalent coupling to LDL.

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Sulfhydryl chemistry and solubility properties of human plasma apolipoprotein B

Cited by 105 publications

References 48 publications

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

A partial cDNA clone for human apolipoprotein B.

Preparation of Nucleoside-LDL-Conjugates for the Study of Cell-Selective Internalization: Stability Characteristics and Receptor Affinity

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