Pulse‐chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2

Wettesten, Margit; Boström, Kristina Bengtsson; Bondjers, Göran; Jarfeldt, Marianne; Norfeldt, Per-Ingvar; Carrella, M; Wiklund, Olov; Borén, Jan; Olofsson, Sven-Olof

doi:10.1111/j.1432-1033.1985.tb08947.x

Cited by 49 publications

(28 citation statements)

References 34 publications

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“…Electrophoresis in SDS-polyacrylamide gels, autoradiography, and determination of the radioactivity in the proteins separated in the gels was carried out as described (29). The culture medium was recovered after the 120-min chase and the secretion of pulse-labeled transferrin was determined.…”

Section: Methodsmentioning

confidence: 99%

Brefeldin A Reversibly Inhibits the Assembly of ApoB Containing Lipoproteins in McA-RH7777 Cells

Rustaeus¹,

Lindberg²,

Borén³

et al. 1995

Journal of Biological Chemistry

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BFA inhibited in a dose dependent way the assembly of apoB-48 very low density lipoprotein (VLDL) but allowed a normal rate of biosynthesis of the apolipoprotein and of the assembly of the dense ("high density lipoprotein (HDL)-like") apoB-48 particle (apoB-48 HDL). The inhibition of the assembly of apoB-48 VLDL occurred at BFA levels that allowed a major secretion of both transferrin and apoB-48 HDL. The assembly of apoB-100 containing lipoproteins was also inhibited by BFA but could be reactivated by a 30 -60 min chase in the absence of BFA, which agreed with the time that was estimated to be needed to restore the secretory pathway (approximately 60 min). Also the assembly of apoB-48 VLDL was reversible. Both apoB-48 and apoB-100 that was labeled in the presence of BFA assembled VLDL after removal of the BFA.Both apoB-100 and apoB-48 were associated with the membrane pellet of the microsomes. Virtually all (122 ؎ 30%) of the membrane associated pulse-labeled apoB-48 remained in the membrane after a 180-min chase in the presence of BFA, compared to only 21 ؎ 2% in normal cells (mean ؎ S.D., n ‫؍‬ 4). The corresponding figures for apoB-100 was 40 ؎ 7% in BFA-treated cells and 9 ؎ 7% in normal cells (mean ؎ S.D., n ‫؍‬ 4).Pulse-chase experiments with BFA offered conditions to selectively follow the turnover of membrane-associated apoB-100. Such experiments indicated that this apoB-100 pool is a precursor to VLDL. Apolipoprotein B (apoB)1 is the major protein component of the triacylglycerol-and cholesterol ester-rich plasma lipoproteins: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins, and low density lipoproteins (LDL).Two forms of apoB exist, referred to as apoB-100 and apoB-48 (1). ApoB-100 consists of 4536 amino acids, while apoB-48 corresponds to the N-terminal 48% of apoB-100. Both proteins are coded for by the same gene (2). The apoB-100 mRNA is converted to mRNA for apoB-48 by an enzymedependent deamination of cytidine in codon 2153, which converts this codon into the signal for translational stop (3-5).In humans, apoB-48 is expressed mainly in the intestine, where it assembles chylomicrons, while apoB-100 is synthesized in the liver and is necessary for the assembly of VLDL particles (6). In contrast, the rat hepatocytes, as well as the rat hepatoma cells McA-RH7777 (7-9) express substantial amounts of both apoB-100 and apoB-48 (7, 10 -12).Recent results (7) from studies in McA-RH7777 cells indicate that during translation/translocation apoB-48 forms a small dense particle (100 Å in diameter), floating as HDL. This small particle appears to acquire the major amount of lipids in a second step and is in this way converted into a VLDL particle. The second step is dependent upon ongoing biosynthesis of proteins other than apoB; moreover, it is stimulated by oleic acid (7).The assembly of apoB-100 VLDL is more complicated (7), and our results indicate that the VLDL particles are assembled at least to a certain extent in relatively close connection with the translation/tra...

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Section: Methodsmentioning

confidence: 99%

Brefeldin A Reversibly Inhibits the Assembly of ApoB Containing Lipoproteins in McA-RH7777 Cells

Rustaeus¹,

Lindberg²,

Borén³

et al. 1995

Journal of Biological Chemistry

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“…There is a temporal delay in the transfer from the ER, which is regulated by the microsomal membrane association of proteins. Albumin, haptoglobin, and transferrin have a half-life of secretion of 30, 40, and 75 min, respectively (41), and it takes ϳ30 min for newly synthesized ApoB 100 to be packaged and released into the circulation (42,43); thus there is a time lag between protein synthesis and appearance in the plasma.…”

Section: Plasma Proteome Dynamicsmentioning

confidence: 99%

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Willard

Rachdaoui

et al. 2012

Molecular & Cellular Proteomics

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Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2 H 2 O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis.2 H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2 H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

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“…Precipitates from all fractions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography (14). ApoB-100 was present in three well separated fractions: the upper 3 ml of the gradient (d Ͻ 1.006 g/ml, corresponding to VLDL), the middle of the gradient (d ϭ 1.006 -1.06 g/ml), and the bottom of the gradient (d ϭ 1.06 -1.21 g/ml).…”

Section: S]methionine and L-[mentioning

confidence: 99%

ADP-ribosylation Factor 1 and Its Activation of Phospholipase D Are Important for the Assembly of Very Low Density Lipoproteins

Asp¹,

Claesson²,

Borén³

et al. 2000

Journal of Biological Chemistry

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thesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10 -15% of the apoB-100 pool was converted to VLDL in a time-and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to Nterminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

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Pulse‐chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2

Cited by 49 publications

References 34 publications

Brefeldin A Reversibly Inhibits the Assembly of ApoB Containing Lipoproteins in McA-RH7777 Cells

Brefeldin A Reversibly Inhibits the Assembly of ApoB Containing Lipoproteins in McA-RH7777 Cells

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

ADP-ribosylation Factor 1 and Its Activation of Phospholipase D Are Important for the Assembly of Very Low Density Lipoproteins

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