Hepatic and intestinal contribution of two forms of apolipoprotein B to plasma lipoprotein fractions in the rat

1981 ◽  
Vol 59 (8) ◽  
pp. 693-699 ◽  
Author(s):  
Charles E. Sparks ◽  
Oleh Hnatiuk ◽  
Julian B. Marsh
Keyword(s):  
Amino Acids ◽  
Apolipoprotein B ◽  
Corn Oil ◽  
Gel Filtration ◽  
Molecular Size ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Apo B ◽  
Triglyceride Rich Lipoprotein

The in vivo incorporation of labeled amino acids into two forms of apolipoprotein B of nascent hepatic, nascent intestinal, and plasma lipoproteins was studied. Using SDS–gel filtration column chromatography rat apolipoprotein B was separated into two proteins of higher (apo Bh) and of lower (apo B1) molecular size and the incorporation of label into each was measured. When livers isolated from fed rats were perfused with 3H-labeled amino acids, radioactivity was incorporated into both forms of apo B of the d < 1.060 fractions (very low (VLDL), intermediate (IDL), and low (LDL) density lipoproteins) with a labeling ratio of apo B1 to apo Bh of 0.8. When mesenteric lymph was collected from corn oil fed rats intraduodenally injected with 3H-labeled amino acids, radioactivity was mainly incorporated into apo B1 of chylomicrons and VLDL with apo B1 to apo Bh, labeling ratios of 14 and 44, respectively. Plasma was isolated 2 h after intraperitoneal injection of 3H-labeled amino acids into chow fed rats and lipoproteins were isolated by sequential density ultracentrifugation. The labeling ratio of apo B1 and apo Bh decreased from 4.2 in VLDL to 0.5 in LDL indicating a progressive enrichment of apo Bh in the LDL fraction. High density lipoproteins (HDL) contained less than 4% of the total labeled apo B and was enriched in apo B1. The results of this study indicate that the liver synthesizes both forms of apo B while the intestine synthesizes almost entirely apo B1. Since both apo B proteins are secreted primarily by the liver into VLDL, the results are consistent with preferential removal of apo B1 during triglyceride-rich lipoprotein catabolism and entry of hepatically derived apo Bh into LDL.

scholarly journals Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins.

1984 ◽  
Vol 159 (2) ◽  
pp. 604-616 ◽  
Author(s):  
I F Rowe ◽  
A K Soutar ◽  
I M Trayner ◽  
M L Baltz ◽  
F C de Beer ◽  
...  
Keyword(s):  
Acute Phase ◽  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Free Form ◽  
Low Density ◽  
Serum Samples ◽  
Acute Phase Serum

Immobilized rabbit and rat C-reactive protein (CRP) were found to selectively bind apolipoprotein B (apoB)-containing lipoproteins (low density lipoprotein, LDL and very low density lipoprotein, VLDL) from whole serum in a manner similar to that previously reported with human CRP. In acute phase human serum the CRP is in a free form, not complexed with lipoprotein or any other macromolecular ligand, and in acute phase serum from most rabbits fed on a normal diet the rabbit CRP was also free. However, in acute phase serum or heparinized plasma from hypercholesterolemic rabbits part or all of the CRP was found by gel filtration and immunoelectrophoretic techniques to be complexed with beta-VLDL, an abnormal apoB-containing plasma lipoprotein present in these animals. The presence of extent in different serum samples of CRP complexed with lipoprotein correlated closely with the serum apoB concentration. The formation of complexes between native, unaggregated rabbit CRP in solution and apoB-containing lipoproteins was readily demonstrable experimentally both with the isolated proteins and in whole serum. In all cases these interactions were calcium-dependent and inhibitable by free phosphoryl choline. The present findings extend earlier work in man and the rabbit and indicate that among the C-reactive proteins from different species, which are structurally highly conserved, the capacity for selective binding to apoB-containing plasma lipoproteins is also a constant feature. These interactions may therefore be related to the in vivo function of CRP in all species and this function may in turn be relevant to pathological conditions, such as atherosclerosis, in which lipoproteins are important.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Molecular Size Distribution of Fibrinogen Derivatives Formed in Vitro and in Vivo: a Chromatographic Study

1976 ◽  
Vol 36 (01) ◽  
pp. 014-026 ◽  
Author(s):  
M. B Donati ◽  
R Verhaeghe ◽  
D. E Culasso ◽  
J Vermylen
Keyword(s):  
Biological Fluids ◽  
Gel Filtration ◽  
Molecular Size ◽  
Gel Chromatography ◽  
Chromatographic Study ◽  
Elution Volume ◽  
Human Fibrinogen ◽  
Elution Pattern

SummaryUsing gel chromatography, fibrinogen derivatives present in purified systems or in biological fluids were separated and partially characterized. Eight groups of fibrinogen derivatives could be separated by gel filtration through 6% agarose in large columns, four with an elution volume smaller and four groups with an elution volume larger than that of fibrinogen. Careful calibration of the column allowed estimation of the diffusion coefficients of some of the derivatives and, thus, comparison with derivatives previously identified. Three, rather than two, groups of intermediate derivatives were observed during the degradation of human fibrinogen by plasmin in vitro or in vivo. One of these had a marked tendency to polymerize.A rather distinct difference in elution pattern was found between plasma obtained during streptokinase administration and from patients with intravascular coagulation.

Variations in the composition of low- and high-density lipoproteins during the acute phase of myocardial infarction.

1988 ◽  
Vol 34 (1) ◽  
pp. 139-140 ◽  
Author(s):  
F Mainard ◽  
Y Madec ◽  
N Robinet
Keyword(s):  
Myocardial Infarction ◽  
Acute Phase ◽  
Apolipoprotein B ◽  
Low Density Lipoproteins ◽  
High Density ◽  
Apolipoprotein A1 ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Apo B ◽  
B Values

Abstract We analyzed correlations between apolipoprotein B (apo B), cholesterol and phospholipids (preponderant lipids) in low-density lipoproteins (LDL) as well as between apolipoprotein A1 (apo A1) and these same lipids in high-density lipoproteins (HDL), during the acute phase of myocardial infarction. In LDL, a very elevated and stable correlation (r) was observed between these parameters, and the coefficients of regression (b) did not differ significantly during the period studied. In HDL, there was a decrease in r and b values from day 1 to day 2, then an increase after day 2. We hypothesize that these disturbances in HDL composition may be due to a greater endocytosis of LDL at day 2, leading to intracellular increase in cholesterol and phospholipids. Part of these lipids could be taken up by HDL molecules, causing a transient overload.

scholarly journals THE ANTICOAGULANT AND ANTILYMPHOMA PROPERTIES OF ARSENIC AZOPROTEINS

1964 ◽  
Vol 120 (3) ◽  
pp. 449-466 ◽  
Author(s):  
J. D. Broome ◽  
John G. Kidd
Keyword(s):  
Amino Acids ◽  
Human Blood ◽  
Inhibitory Action ◽  
Anticoagulant Activity ◽  
Molecular Size ◽  
Structural Requirements ◽  
Time Required

Experiments given in this paper have shown that 4-arsonophenylazoproteins possess marked anticoagulant activity both in vivo and in vitro. Mice and rabbits given moderate amounts of the arsenic azoprotein, for example, often bled to death from injuries that proved trivial in control animals, and their blood remained liquid during many hours' postmortem even when left in contact with transected tissues, fibrinolysis having no part in the outcome. So, too, the addition of minute amounts of 4-arsonophenylazoprotein to plasma procured from citrated rabbit or human blood greatly prolonged the time required for clotting after recalcification. Other arsenic-containing compounds,—for example, those in which arsenic See PDF for Structure was joined to amino acids or peptides through the azo linkage, or to proteins through couplings other than the azo linkage,—were largely devoid of anticoagulant and antilymphoma effects. The findings as a whole show clearly that the structural requirements for anticoagulant and antilymphoma effects are: (a) possession of negatively charged arsonic or arsinoso groups, (b) large molecular size (protein), and (c) linkage of arsenic-containing groups to protein through the azo bond. Two acidic azoproteins that were devoid of arsenic,—namely 4-carboxyphenylazoprotein and 4-sulfonophenylazoprotein,—were also found to have marked anticoagulant effects in vitro, but they had no inhibitory action against cells of Lymphoma 6C3HED in vivo, even when they were given to mice in maximum tolerated amounts. The essential part played by arsenic in the antilymphoma activity of arsenic azoproteins was further emphasized by the action of dimercaprol (BAL) in preventing the antilymphoma effects of 4-arsonophenylazoprotein on Lymphoma 6C3HED cells in vivo. In an associated paper the anticoagulant and antilymphoma effects of 4-arsonophenylazoproteins are studied further, and consideration is given to the ways in which these effects may be brought about.

scholarly journals Species Differences in the Proportion of Plasma Lipoprotein Lipid Carried by High-Density Lipoproteins Influence the Distribution of Free and Liposomal Nystatin in Human, Dog, and Rat Plasma

1999 ◽  
Vol 43 (6) ◽  
pp. 1424-1428 ◽  
Author(s):  
Manisha Ramaswamy ◽  
Thomas L. Wallace ◽  
Paul A. Cossum ◽  
Kishor M. Wasan
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Rat Plasma ◽  
High Density ◽  
Plasma Lipoprotein ◽  
Lipoprotein Cholesterol ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Lipoprotein Lipid ◽  
Triglyceride Rich Lipoprotein

ABSTRACT The objective of this study was an interspecies comparison of free nystatin (NYS) and liposomal NYS (Nyotran) distribution in plasma. NYS and liposomal NYS at concentrations of 5, 10, and 20 μg of NYS/ml were incubated in human, dog, and rat plasma for 5, 60, and 180 min at 37°C. Following these incubations, plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein, low-density lipoprotein, and lipoprotein-deficient plasma (LPDP) fractions by density-gradient ultracentrifugation, and each fraction was assayed for NYS by high-pressure liquid chromatography. Total plasma and lipoprotein cholesterol, triglyceride, and protein concentrations in each human, dog, or rat plasma sample were determined by enzymatic assays. When NYS and liposomal NYS were incubated in human, dog, or rat plasma, the majority of the NYS was recovered in the LPDP fraction. For the 5- and 60-min incubation times for all plasmas measured, a significantly greater percentage of NYS was recovered in the lipoprotein fraction (primarily HDL) following the incubation of liposomal NYS than following the incubation of NYS. There was a significant correlation between the lipoprotein lipid and protein profiles in human, dog, and rat plasmas and the distribution of NYS and liposomal NYS in plasma. In particular, differences in the proportion of plasma lipoprotein cholesterol, triglyceride, and apolar lipids (cholesteryl ester and triglycerides) carried by HDL influenced the distribution of NYS and liposomal NYS within plasmas of different species. These findings suggest that the distribution of NYS among plasma lipoproteins of different species is defined by the proportion of lipid carried by HDL, and this is possibly an important consideration when evaluating the pharmacokinetics, toxicities, and activities of these compounds following administration to different animal species.

Tunicamycin induces ubiquitination and degradation of apolipoprotein B in HepG2 cells

2001 ◽  
Vol 353 (3) ◽  
pp. 493-501 ◽  
Author(s):  
Wei LIAO ◽  
Lawrence CHAN
Keyword(s):  
Apolipoprotein B ◽  
Hepg2 Cells ◽  
Proteasome Inhibitor ◽  
Plasma Lipoproteins ◽  
Apo B ◽  
Net Production ◽  
Lipoprotein Particles ◽  
Essential Component ◽  
Intracellular Degradation

Apolipoprotein (apo) B-100 is an essential component of atherogenic plasma lipoproteins. Previous studies have demonstrated that the production of apoB-100 is regulated largely by intracellular degradation at both the co-translational and post-translational levels and that proteasome-mediated and non-proteasome-mediated pathways are involved in this process. ApoB-100 is a glycoprotein. The present study was undertaken to address the question of whether the inhibition of N-linked glycosylation with tunicamycin would interfere with apoB-100 production. We demonstrated that the treatment of HepG2 cells with tunicamycin decreased the net production of apoB-100 by enhancing co-translational degradation of the protein. This effect of tunicamycin was partly prevented by lactacystin, a specific proteasome inhibitor. Because lactacystin only partly reversed the effects of tunicamycin on apoB biogenesis, tunicamycin seemed also to induce apoB co-translational degradation in HepG2 cells by one or more non-proteasomal pathways. Furthermore, tunicamycin increased apoB ubiquitination approx. 4-fold. The proportion of the newly synthesized apoB-100 that was secreted and incorporated into the nascent lipoprotein particles was unaffected by tunicamycin. Thus the tunicamycin-mediated inhibition of N-linked glycosylation interferes with the production of apoB-100 that is mediated by both proteasomal and non-proteasomal pathways.

Reduction in VLDL, but not HDL, in plasma of rats deficient in choline

1990 ◽  
Vol 68 (2) ◽  
pp. 552-558 ◽  
Author(s):  
Zemin Yao ◽  
Dennis E. Vance
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Density ◽  
Plasma Lipoproteins ◽  
Male Rats ◽  
High Density Lipoproteins ◽  
Choline Deficiency ◽  
Deficient Diet ◽  
Apo B

We have analyzed plasma lipoprotein levels in young male rats fed a choline-deficient diet for 3 days. We confirmed previous studies that choline deficiency promotes 6.5-fold accumulation of triacylgycerol in the liver (23.9 ± 6.0 versus 3.69 ± 0.92 μmol/g liver) and reduction of triacylglycerol concentration in plasma by 60% (0.17 ± 0.04 versus 0.46 ± 0.10μmol/mL plasma). Agarose gel electrophoresis showed that the plasma very low density lipoprotein (VLDL) levels were reduced in choline-deficient rats, but the concentration of plasma high density lipoproteins (HDL) was not affected. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of fractionated plasma lipoproteins revealed that the concentrations of apolipoproteins (apo) BH, BL, and E in VLDL from choline-deficient rats were 37.1, 11.0, and 37.2% of normal levels, respectively. In contrast, the amount of apo A-I, the major one in HDL, was almost unchanged. Correspondingly, there were decreased lipid (mainly phosphatidylcholine and triacylglycerol) levels in VLDL from choline-deficient rats, but no change in the levels of phosphatidylcholine, cholesterol, and cholesterol ester in HDL. There were similar levels of apo B and E (components of VLDL) in homogenates of livers from normal and choline-deficient rats, as determined by immunoblotting. These results support the hypothesis that choline deficiency causes reduction of VLDL, but not HDL, levels in plasma as a consequence of impaired hepatic VLDL secretion.Key words: choline deficiency, very low and high density lipoproteins, apolipoproteins, rat plasma.

scholarly journals Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

1997 ◽  
Vol 8 (4) ◽  
pp. 637-645 ◽  
Author(s):  
J Ozols ◽  
J M Caron
Keyword(s):  
Amino Acids ◽  
Structure Analysis ◽  
Structural Information ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Protein Structure Analysis ◽  
Free System ◽  
Alpha Tubulin ◽  
Companion Article

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.

scholarly journals Altered Oligomerization Properties of N316 Mutants of Escherichia coli TyrR

2008 ◽  
Vol 190 (24) ◽  
pp. 8238-8243 ◽  
Author(s):  
Takashi Koyanagi ◽  
Takane Katayama ◽  
Hideyuki Suzuki ◽  
Hidehiko Kumagai
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Gel Filtration ◽  
Aromatic Amino Acids ◽  
Transcriptional Regulator ◽  
Wild Type ◽  
Oligomer Formation ◽  
Regulatory Properties

ABSTRACT The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316D and TyrRN316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316D and TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.

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