FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2058588045> ?p ?o ?g. }

• W2058588045 endingPage "10513" @default.
• W2058588045 startingPage "10506" @default.
• W2058588045 abstract "We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [3H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 ± 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in thed < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum. We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [3H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 ± 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in thed < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum. apolipoprotein endoplasmic reticulum very low density lipoprotein(s) high density lipoprotein(s) polyacrylamide gel electrophoresis Immunoelectron microscopy studies have shown that apolipoprotein (apo)1 B is present in the rough endoplasmic reticulum (ER), but very low density lipoprotein (VLDL)-sized particles are not (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar). VLDL particles with immunoreactive apoB first appeared in the smooth termini of the rough ER; the smooth ER contained VLDL-sized particles without immunoreactive apoB (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar). Based on these results, a two-step model for the assembly of VLDL was proposed. Dynamic evidence for this model was obtained by pulse-chase studies of apoB-100 and apoB-48 (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 3.Swift L.L. J. Lipid Res. 1995; 36: 395-406Abstract Full Text PDF PubMed Google Scholar). The first step occurs during the translation of apoB and gives rise to a partially lipidated form of apoB (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 4.Borén J. Graham L. Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar). In the case of apoB-100, this partially lipidated particle appeared to be loosely associated with the ER membrane (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the case of apoB-48, a particle resembling high density lipoprotein (HDL) has been identified. The secretion of this dense, apoB-48-containing, HDL-like lipoprotein varied inversely with that of VLDL (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Therefore, we hypothesized that this particle is a precursor of apoB-48 VLDL (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). A second VLDL precursor was identified as an apoB-free “lipid droplet” in the smooth ER (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar, 6.Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar). The assembly of both precursors is dependent on the microsomal triglyceride transfer protein (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 7.Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Borén J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,8.Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). The mechanism for the second step, fusion of the two precursors (1.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar), is less well understood. We have demonstrated that brefeldin A inhibits the major lipidation of apoB (9.Rustaeus S. Lindberg K. Borén J. Olofsson S.-O. J. Biol. Chem. 1995; 270: 28879-28886Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However the exact localization of the brefeldin A-sensitive mechanism in the assembly pathway remains to be elucidated. Cotranslational or early post-translational degradation of apoB is important in regulating the amount of apoB that passes through the first step (10.Zhou M. Wu X. Huang L.S. Ginsberg H.N. J. Biol. Chem. 1995; 270: 25220-25224Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11.Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). This degradation involves ubiquitination and proteasomes (11.Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Recent results indicate that apoB is completely translocated to the lumen of the ER (12.Shelness G.S. Morris-Rogers K.C. Ingram M.F. J. Biol. Chem. 1994; 269: 9310-9318Abstract Full Text PDF PubMed Google Scholar), suggesting that the early post-translational degradation follows the pathway described for misfolded proteins (i.e. the protein is retracted through the translocation channel) (13.Cresswell P. Hughes E.A. Curr. Biol. 1997; 7: 552-555Abstract Full Text Full Text PDF PubMed Google Scholar). However, there is strong evidence that the degradation involves nascent as well as full-length apoB chains (14.Zhou M. Fisher E.A. Ginsberg H.N. J. Biol. Chem. 1998; 273: 24649-24653Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 15.Liao W. Yeung S.-C.J. Chan L. J. Biol. Chem. 1998; 273: 27225-27230Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), suggesting that this proteasomal degradation may also involve other pathways (for reviews, see Refs. 16.Ginsberg H.N. Clin. Exp. Pharmacol. Physiol. 1997; 24: A29-A32Crossref PubMed Scopus (49) Google Scholar and 17.Olofsson S.-O. Asp L. Borén J. Curr. Opin. Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (188) Google Scholar). Studies of VLDL assembly have been hampered by the fact that much of the apoB present in the cell remains associated with the microsomal membrane after carbonate extraction of the luminal proteins and therefore cannot be analyzed. Recently, we developed a procedure that extracts virtually all of the apoB-100 from the microsomal membranes without releasing integral membrane proteins (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this study, we used this new extraction procedure in a series of experiments to analyze the assembly of apoB-48 VLDL. Eagle's minimum essential medium, nonessential amino acids, glutamine, penicillin, and streptomycin were obtained from ICN Biomedicals (Costa Mesa, CA). Fetal calf serum was from Biochrom KG (Berlin, Germany) and brefeldin A from Epicenter Technologies (Madison, WI). Methionine, fatty acid-free bovine serum albumin, sodium pyruvate, disodium carbonate, sodium hydrogen carbonate, phenylmethylsulfonyl fluoride, pepstatin A, and leupeptin were from Sigma. Rabbit immunoglobulin was from Dako (Glostrup, Denmark), and rabbit anti-rat transferrin IgG was from Organon Teknika (West Chester, PA). Trasylol (aprotinin) was from Bayer (Leverkusen, Germany). Immunoprecipitin and Eagle's minimum essential medium without methionine were from Life Technologies, Inc. N-Acetyl-Leu-Leu-norleucinal as well as enzymatic assays for the determination of phospholipids or triglycerides were from Boehringer Mannheim. Amplify, [35S]methionine/cysteine mix, Rainbow protein molecular weight markers, and the ECL Western blotting analysis system were from Amersham Pharmacia Biotech, and Ready-Safe was from Beckman (Fullerton, CA). All chemicals used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase-conjugated goat anti-rabbit and rabbit anti-mouse were from Bio-Rad (Hercules, CA). Blue-stabilized substrate for alkaline phosphatase and trypsin (sequencing grade) were from Promega (Milwaukee, WI). Cyanogen bromide-activated Sepharose 4B was from Amersham Pharmacia Biotech, and α-cyano-4-OH cinnamic acid was from Aldrich (Milwaukee, WI). Antibodies to chaperones (binding protein, protein disulfide isomerase, glucose regulatory protein 94, and calreticulin) were purchased from Affinity BioReagents (Golden, CO). McA-RH7777 cells were cultured as described previously (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar) in Eagle's minimum essential medium containing 20% fetal calf serum, 1.6 mm glutamine, 8.0 mmNaHCO3, 1.6 mm sodium pyruvate, 140 mg/ml streptomycin, 140 IU/ml penicillin, and 60 mg/ml nonessential amino acids in 5% CO2 at 37 °C. The cultures were split twice a week and fed daily. The cells were pulse labeled and chased as described (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Cells and the microsomal fraction were isolated as described (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar). The luminal content of the vesicles was separated from the vesicle membranes by the sodium carbonate method (19.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar), as modified (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). The following protease inhibitors were used: 0.1 mmleupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A, 5 mm N-acetyl-Leu-Leu-norleucinal, and aprotinin (100 kallekrein-inhibitory units/ml). In some experiments, the luminal content of the microsomal vesicles was extracted with the deoxycholate/carbonate procedure described recently by Rustaeuset al. (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The total microsomal fraction was obtained (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar) and fractionated on a sucrose gradient. A 3.8-ml linear sucrose gradient (32.5–40%, w/v) was layered on a cushion of 0.5 ml of 65% sucrose (w/v), and the sample was layered on top of the gradient. All solutions contained 3 mm imidazole, pH 7.4, with the same protease inhibitors as used for metabolic labeling. The gradients were centrifuged in a Beckman Vti-65.2 vertical rotor at 50,000 rpm for 3 h at 12 °C. The gradient was unloaded from the bottom into 22 fractions. NADPH cytochrome c reductase and galactosyl transferase were used as marker enzymes for the ER and the Golgi apparatus, respectively (20.Borén J. Wettesten M. Sjöberg A. Thorlin T. Bondjers G. Wiklund O. Olofsson S.-O. J. Biol. Chem. 1990; 265: 10556-10564Abstract Full Text PDF PubMed Google Scholar). Gradient fractions were also assayed by Western blot for calnexin, a marker for the rough ER. Lipoproteins in the microsomal lumen or in the medium were separated by sucrose gradient ultracentrifugation (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). The gradient was formed by layering, from the bottom of the tube, 2 ml of 49% sucrose, 2 ml of 25% sucrose, 5 ml of the sample in 12.5% sucrose (the sucrose was in phosphate-buffered saline), and 3 ml of phosphate-buffered saline. All solutions contained 0.1 mm leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A, 5 mm N-acetyl-Leu-Leu-norleucinal, aprotinin (100 kallekrein-inhibitory units/ml), and 0.5 mm EDTA. The gradients were centrifuged in a Beckman SW40 rotor at 35,000 rpm for 65 h at 12 °C and unloaded from the bottom of the tube into 12–13 fractions. ApoB was immunoprecipitated from the cells, medium, and sucrose gradient fractions as described (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar, 21.Wettesten M. Boström K. Bondjers G. Jarfeldt M. Norfeldt P.-I. Carrella M. Wiklund O. Borén J. Olofsson S.-O. Eur. J. Biochem. 1985; 149: 461-466Crossref PubMed Scopus (32) Google Scholar). Immunoaffinity chromatography of apoB-containing fractions extracted from the microsomes or present in the culture medium was carried out as described (18.Boström K. Borén J. Wettesten M. Sjöberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.-O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE, autoradiography, and determination of the radioactivity in proteins separated in the gels were performed as detailed elsewhere (21.Wettesten M. Boström K. Bondjers G. Jarfeldt M. Norfeldt P.-I. Carrella M. Wiklund O. Borén J. Olofsson S.-O. Eur. J. Biochem. 1985; 149: 461-466Crossref PubMed Scopus (32) Google Scholar). Lipids from McA-RH7777 cells were extracted as described by Olegård and Svennerholm (22.Olegård R. Svennerholm L. Acta Paediatr. Scand. 1970; 59: 637-647Crossref PubMed Scopus (108) Google Scholar) with slight modifications (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Phosphatidylcholine and triglycerides were separated by thin layer chromatography with chloroform:methanol:water (65:25:4 v/v) followed by petroleumether:diethylether:acetic acid (80:20:1 v/v). The spots were visualized by iodine, scraped off, and extracted with 1 ml of chloroform:methanol 1:2 (phosphatidylcholine) or chloroform (triglycerides). The extracted lipids were dried under nitrogen in a conical tube, solubilized in 20 μl of ethanol, and analyzed by enzymatic assays. As standards, pure phosphatidylcholine and triglycerides were chromatographed and processed in parallel with the sample. The recovery of triglycerides and phosphatidylcholine during the chromatography and extraction steps was 94 ± 16% (mean ± S.D.; n = 5) and 65 ± 4% (n = 5). The recovery of triglycerides was also tested with radioactive tracer added to the cell homogenate; this experiment showed a recovery of 94 ± 8% (n = 4). The intra-assay variation was 4.5% for triglycerides and 8.1% for phosphatidylcholine. Phosphatidylcholine and triglycerides were radiolabeled by incubating the cells for various periods with [3H]glycerol (0.6 μCi/ml of culture medium). Cells were extracted, phosphatidylcholine and triglycerides were separated as described above, and specific radioactivity was determined (dpm/mg). In some experiments, apoB-containing lipoproteins were isolated by immunoaffinity chromatography from the luminal content or the medium. During the extraction of the lipids from these fractions, unlabeled phosphatidylcholine and triglycerides were added as carriers. The lipids were separated as described above, and the spots corresponding to triglycerides and phosphatidylcholine were scraped into scintillation vials; 1 ml of cyclohexane was added, and the radioactivity was determined in the presence of Ready-Safe scintillation mixture. Proteins associated with microsomal lipoproteins were isolated and identified as follows. Rat liver microsomes were isolated (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar), and the luminal content was extracted with sodium carbonate (19.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar). The extract (6 ml) was overlayered with 29 ml of phosphate-buffered saline (8 mm disodium hydrogen phosphate, 1.5 mmpotassium dihydrogen phosphate, 137 mm sodium chloride, and 2.7 mm potassium chloride, pH 7.4, d = 1.006 g/ml). After centrifugation for 22 h at 40,000 rpm in a Beckman Ti-60 rotor at 4 °C, the gradients were fractionated from the top. The upper one-third of the tube (d < 1.006 g/ml) was collected. Pooled fractions of this supernatant (corresponding to three to five rat livers) were loaded onto a Mono Q column equilibrated with 50 mm Tris-HCl, pH 7.8, with 300 mm sucrose, 1 mm EDTA, 2 mmdeoxycholate, 0.5% Triton X-100, 6 m urea, and 40 mm sodium carbonate. The column was eluted with a linear gradient of sodium chloride (0–250 mm) at a flow rate of 0.5 ml/min. Fractions (0.5 ml) were collected, and the proteins in each fraction were separated by SDS-PAGE on 10% gels. Gels were stained with silver. Fractions containing the same protein patterns were combined, concentrated, and subjected to SDS-PAGE on 3–15% gradient gels. The gels were stained with Coomassie Brilliant Blue, and the bands were cut out, destained with 50 μl of a mixture of 50% ammonium bicarbonate (25 mm) and 50% acetonitrile, dried, and digested for 15 min with 0.1–0.2 mg of trypsin in 20 μl of 50% ammonium bicarbonate (25 mm) and 50% acetonitrile. Ammonium bicarbonate (25 mm, pH 8) was added to cover the gels, and incubation was continued for 12 h at 37 °C. Fragments were extracted with 10–50 μl of a mixture of 75% acetonitrile and 5% trifluoroacetic acid (in water). Mass spectra were obtained on a TofSpec-E time-of-flight mass spectrometer (Micromass; Manchester, UK) equipped with a time-lagged focusing unit; TOF2UI version 3.4 was used for data collection and OPUS version 3.4 for data analysis. α-Cyano-4-OH cinnamic acid (10 mg/ml in water/acetonitrile, 50/50, v/v) was used as matrix without further purification. The α-cyano-4-OH cinnamic acid solution (0.5 ml) was mixed with the gel extract (0.5 ml) on the target and allowed to dry at room temperature. Spectra were collected in reflectron mode at an accelerating voltage of 20 kV with a 600-ns delayed extraction and a pulse of approximately 2.4 kV. Approximately 200 nitrogen laser pulses (3 ns, 337 nm) were carried out on each sample. For external calibration, a mixture of ACTH and angiotensin II was used (protonated 2465.2 and 1046.5, respectively). The peptide mass fingerprinting software program MS-Fit was run over the Internet. Monoisotopic masses were used for the searches; mass tolerance was ± 200 ppm. To determine the effects of deoxycholate/carbonate extraction (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) on integral microsomal proteins, we first generated a monoclonal antibody against riboforin. BALB/c mice were immunized with solubilized microsomal membrane proteins from rat liver (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Positive hybridomas were identified by enzyme-linked immunosorbent assay with the antigen (solubilized rat liver microsomes) and analyzed by Western blot of solubilized rat liver microsomes. One hybridoma reacted with a 60-kDa protein and was recloned to monoclonality. The immunoglobulins were isolated from the hybridoma culture medium and coupled to cyanogen bromide-activated Sepharose 4B as recommended by the manufacturer (Amersham Pharmacia Biotech) and used for immunoadsorption experiments (23.Andersson M. Wettesten M. Borén J. Magnusson A. Sjöberg A. Rustaeus S. Olofsson S.-O. J. Lipid Res. 1994; 35: 535-545Abstract Full Text PDF PubMed Google Scholar). Using this immunoadsorbent, we recovered the 60-kDa protein that reacted with the monoclonal antibody. This protein was cut out of the Coomassie-stained gel, digested with trypsin, and analyzed by mass spectrometry as described above. A data search identified the protein as rat riboforin. In contrast to ordinary sodium carbonate extraction, the deoxycholate/carbonate procedure (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) extracted virtually all of the apoB-48 (93 ± 1%; n = 5) from the microsomes. As judged from immunoblot studies of calnexin (see Ref. 5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and riboforin (Fig. 1 A) deoxycholate/carbonate extraction did not release integral membrane proteins. Upon gradient ultracentrifugation, the major amount of apoB-48 extracted by the deoxycholate/carbonate method migrated in the HDL density range (Fig. 1 B). Using a modified gradient, we compared the densities of secreted apoB-48 and deoxycholate/carbonate-extracted apoB-48. The major amount of the secreted apoB-48 was present in the VLDL and the HDL density regions, as described previously (2.Borén J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). ApoB-48 that banded in the HDL density region (apoB-48 HDL) migrated in the same position as the apoB-48 extracted from the microsomes (Fig. 1 C); we will refer to this form of apoB-48 as intracellular apoB-48 HDL. Thus, intracellular apoB-48 HDL and secreted apoB-48 HDL have very similar buoyant densities, and each migrated in the gradient in the expected position for a lipoprotein (in comparison with a nonlipidated protein of similar molecular weight) (Fig. 1 C, III). Thus, the membrane-associated apoB-48, like apoB-100 (5.Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), was extracted from the microsomes as a tentative lipoprotein. To determine if intracellular apoB (both B-100 and B-48)-containing HDL is associated with lipids and contains a lipid core, we began by estimating the incubation time needed to obtain steady-state labeling of the phosphatidylcholine and triglyceride pools of the cell. The cells were incubated with [3H]glycerol (0.6 μCi/ml of culture medium) for 0, 1, 2, 5, 8, and 22 h. After each incubation, phosphatidylcholine and triglycerides were isolated, and the specific radioactivity (dpm/mg) was determined. Triglycerides reached a plateau after 8 h; phosphatidylcholine plateaued between 1 and 8 h, after which the specific radioactivity decreased (data not shown). We therefore incubated the cells for 8 h with [3H]glycerol (0.6 μCi/ml culture of medium). The specific radioactivities of the total phosphatidylcholine (4,025 dpm/μg of lipid) and triglyceride (2,044 dpm/μg of lipid) pools of the cell were determined. The apoB-containing lipoproteins in the HDL density region of the deoxycholate/carbonate extract of the microsomes were isolated by immunoaffinity chromatography, and the radioactivity in phosphatidylcholine and triglycerides was determined. Assuming that the specific radioactivity of the glycerolipids in this apoB fraction was the same as that of the total cell, we estimated the weight ratio between triglycerides and phosphatidylcholine in intracellular apoB HDL to be 0.85 ± 0.15 (n = 3; molar ratio, 0.70 ± 0.09), indicating less triglyceride than phospholipid. Thus, intracellular apoB HDL has an immature lipid core. The labeled cells were also chased for 2 h, and apoB HDL in the medium was isolated by gradient ultracentrifugation followed by immunoaffinity chromatography. Analyzed as described above, the weight ratio between triglycerides and phosphatidylcholine was 0.27 ± 0.10 (n = 3; molar ratio 0.22 ± 0.08), indicating that this particle has a lipid core. Next, we performed pulse-chase experiments to follow the turnover of intracellular apoB-48 HDL and correlated the findings with VLDL assembly and the appearance of apoB-48 HDL in the medium. The cells were pulse labeled with [35S]methionine/cysteine for 10 min and chased for 0–120 min. Radioactive apoB-48 was first seen in the microsomal intracellular apoB-48 HDL (Fig.2). Not until maximal apoB-48 radioactivity was reached in this fraction did any significant amount of apoB-48 radioactivity appear in the VLDL fraction. In fact, the decrease in the apoB-48 radioactivity which followed this maximum accounted for the increased radioactivity in apoB-48 VLDL and in secreted apoB-48 HDL. To determine the length of apoB required for VLDL assembly, we performed pulse-chase studies of nascent apoB chains. To obtain a continuous series of apoB polypeptides of different lengths which could be tested in the assembly process, we truncated apoB with cycloheximide, detached the nascent polypeptides from the ribosomes with puromycin, and chased them through the secretory pathway of the cell into the medium (4.Borén J. Graham L. Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar). The cells were pulse labeled for 10 min and chased for 0–30 min. After each chase period, the cells were treated with cycloheximide and puromycin and then chased for another 180 min in the presence of cycloheximide and puromycin to allow the nascent chains to form lipoproteins and be secreted into the medium. The medium (containing full-length apoB-100/48 as well as the nascent apoB polypeptides that were released into the secretory pathway and secreted during the 180-min chase) was subjected to gradient ultracentrifugation. ApoB was recovered from each fract" @default.
• W2058588045 created "2016-06-24" @default.
• W2058588045 creator A5013018709 @default.
• W2058588045 creator A5018230960 @default.
• W2058588045 creator A5022381872 @default.
• W2058588045 creator A5046743882 @default.
• W2058588045 creator A5064345565 @default.
• W2058588045 creator A5066716862 @default.
• W2058588045 creator A5085421806 @default.
• W2058588045 date "2000-04-01" @default.
• W2058588045 modified "2023-09-30" @default.
• W2058588045 title "The Assembly and Secretion of Apolipoprotein B-48-containing Very Low Density Lipoproteins in McA-RH7777 Cells" @default.
• W2058588045 cites W11746928 @default.
• W2058588045 cites W1526579847 @default.
• W2058588045 cites W1541084827 @default.
• W2058588045 cites W1551374056 @default.
• W2058588045 cites W1557907146 @default.
• W2058588045 cites W1921601722 @default.
• W2058588045 cites W1965460266 @default.
• W2058588045 cites W1977820944 @default.
• W2058588045 cites W1977941641 @default.
• W2058588045 cites W1985826486 @default.
• W2058588045 cites W1992992449 @default.
• W2058588045 cites W1997826161 @default.
• W2058588045 cites W1999233154 @default.
• W2058588045 cites W2008688211 @default.
• W2058588045 cites W2018894673 @default.
• W2058588045 cites W2018995123 @default.
• W2058588045 cites W2025872868 @default.
• W2058588045 cites W2040661765 @default.
• W2058588045 cites W2052008556 @default.
• W2058588045 cites W2065656920 @default.
• W2058588045 cites W2069663235 @default.
• W2058588045 cites W2089374333 @default.
• W2058588045 cites W2122433263 @default.
• W2058588045 cites W2122865563 @default.
• W2058588045 cites W2129386115 @default.
• W2058588045 cites W2138623532 @default.
• W2058588045 cites W2139766250 @default.
• W2058588045 cites W2157680057 @default.
• W2058588045 cites W2160107586 @default.
• W2058588045 cites W2166851392 @default.
• W2058588045 cites W2171457437 @default.
• W2058588045 cites W2375812937 @default.
• W2058588045 cites W261742487 @default.
• W2058588045 doi "https://doi.org/10.1074/jbc.275.14.10506" @default.
• W2058588045 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10744742" @default.
• W2058588045 hasPublicationYear "2000" @default.
• W2058588045 type Work @default.
• W2058588045 sameAs 2058588045 @default.
• W2058588045 citedByCount "74" @default.
• W2058588045 countsByYear W20585880452012 @default.
• W2058588045 countsByYear W20585880452013 @default.
• W2058588045 countsByYear W20585880452016 @default.
• W2058588045 countsByYear W20585880452021 @default.
• W2058588045 countsByYear W20585880452022 @default.
• W2058588045 countsByYear W20585880452023 @default.
• W2058588045 crossrefType "journal-article" @default.
• W2058588045 hasAuthorship W2058588045A5013018709 @default.
• W2058588045 hasAuthorship W2058588045A5018230960 @default.
• W2058588045 hasAuthorship W2058588045A5022381872 @default.
• W2058588045 hasAuthorship W2058588045A5046743882 @default.
• W2058588045 hasAuthorship W2058588045A5064345565 @default.
• W2058588045 hasAuthorship W2058588045A5066716862 @default.
• W2058588045 hasAuthorship W2058588045A5085421806 @default.
• W2058588045 hasBestOaLocation W20585880451 @default.
• W2058588045 hasConcept C126322002 @default.
• W2058588045 hasConcept C134018914 @default.
• W2058588045 hasConcept C185592680 @default.
• W2058588045 hasConcept C2778163477 @default.
• W2058588045 hasConcept C2780072125 @default.
• W2058588045 hasConcept C49039625 @default.
• W2058588045 hasConcept C62746215 @default.
• W2058588045 hasConcept C71924100 @default.
• W2058588045 hasConcept C8243546 @default.
• W2058588045 hasConceptScore W2058588045C126322002 @default.
• W2058588045 hasConceptScore W2058588045C134018914 @default.
• W2058588045 hasConceptScore W2058588045C185592680 @default.
• W2058588045 hasConceptScore W2058588045C2778163477 @default.
• W2058588045 hasConceptScore W2058588045C2780072125 @default.
• W2058588045 hasConceptScore W2058588045C49039625 @default.
• W2058588045 hasConceptScore W2058588045C62746215 @default.
• W2058588045 hasConceptScore W2058588045C71924100 @default.
• W2058588045 hasConceptScore W2058588045C8243546 @default.
• W2058588045 hasIssue "14" @default.
• W2058588045 hasLocation W20585880451 @default.
• W2058588045 hasOpenAccess W2058588045 @default.
• W2058588045 hasPrimaryLocation W20585880451 @default.
• W2058588045 hasRelatedWork W1999568358 @default.
• W2058588045 hasRelatedWork W2005812619 @default.
• W2058588045 hasRelatedWork W2019231080 @default.
• W2058588045 hasRelatedWork W2026539275 @default.
• W2058588045 hasRelatedWork W2039250890 @default.
• W2058588045 hasRelatedWork W2042275719 @default.
• W2058588045 hasRelatedWork W2081064907 @default.
• W2058588045 hasRelatedWork W2117255531 @default.
• W2058588045 hasRelatedWork W2129790693 @default.
• W2058588045 hasRelatedWork W2402054150 @default.

• next