scholarly journals Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.

1976 ◽  
Vol 69 (2) ◽  
pp. 241-263 ◽  
Author(s):  
C A Alexander ◽  
R L Hamilton ◽  
R J Havel
Keyword(s):  
Golgi Apparatus ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Antigenic Determinants ◽  
Secretory Vesicles ◽  
Low Density ◽  
Bile Canaliculi ◽  
Very Low Density Lipoproteins ◽  
Rough Er ◽  
Space Of Disse

Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.

Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus

1977 ◽  
Vol 55 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Peter J. Dolphin ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Secretory Vesicles ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Experimental Conditions ◽  
Apo B

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation. The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.

scholarly journals Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus.

1985 ◽  
Vol 100 (5) ◽  
pp. 1558-1569 ◽  
Author(s):  
C A Hornick ◽  
R L Hamilton ◽  
E Spaziani ◽  
G H Enders ◽  
R J Havel
Keyword(s):  
Golgi Apparatus ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Endocytic Pathway ◽  
Multivesicular Bodies ◽  
Secretory Vesicles ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Isolation And Characterization

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor-mediated endocytosis. MVBs also contained numerous small vesicles, 0.05-0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver

1976 ◽  
Vol 54 (7) ◽  
pp. 617-628 ◽  
Author(s):  
A. Christine Nestruck ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Density Lipoproteins ◽  
Polyacrylamide Gel ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Relationship Analysis ◽  
Discontinuous Sucrose Gradient

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor–product relationship. Analysis of the apoproteins of the Golgi VLDL by polyacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF3, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1 showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent.The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.

Subcellular Localization of “B” Apoprotein of Plasma Lipoproteins in Rat Liver

1974 ◽  
Vol 32 ◽  
pp. 238-239
Author(s):  
Cheryl Ann Alexander ◽  
Robert L. Hamilton ◽  
Richard J. Havel
Keyword(s):  
Horseradish Peroxidase ◽  
Sodium Phosphate ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Step Method ◽  
Very Low Density Lipoproteins ◽  
Sodium Phosphate Buffer ◽  
Mammalian Liver ◽  
Long Evans

The B apoprotein is thought to be essential for the synthesis and secretion of triglyceriderich lipoproteins from mammalian liver. Antibodies were prepared against rat low density lipoproteins (LDL, density 1.025-1.045) and very low density lipoproteins (VLDL). By Ouchterlony immunodiffusion and Immunoelectrophoresis, the major determinant shared by these antisera had the characteristics of the B apoprotein. Fab fragments were prepared by the method of Porter and conjugated to horseradish peroxidase (HRP) by the two step method of Avrameas and Ternynck. Male Long-Evans rats (250-300 gm) were fasted overnight, anesthetized with Brevital (Lilly), and perfused through the portal vein with fresh 4% formaldehyde in 0.135 M sodium phosphate buffer.

scholarly journals Characterization of plasma lipoproteins separated and purified by agarose-column chromatography

1974 ◽  
Vol 139 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Lawrence L. Rudel ◽  
Jason A. Lee ◽  
Manford D. Morris ◽  
James M. Felts
Keyword(s):  
Human Plasma ◽  
Column Chromatography ◽  
Low Density Lipoproteins ◽  
High Density ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Simple Method ◽  
Lipoprotein Class

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.

Measurement of plasma lipoproteins by electrophoresis on polyacrylamide gel.

1977 ◽  
Vol 23 (10) ◽  
pp. 1826-1833 ◽  
Author(s):  
N Muñiz
Keyword(s):  
Clinical Application ◽  
Classical Method ◽  
Correlation Coefficients ◽  
Low Density Lipoproteins ◽  
Polyacrylamide Gel ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Densitometric Analysis ◽  
Routine Clinical Application

Abstract Results of densitometric analysis of lipoprotein peaks after electrophoresis on polyacrylamide gel and values obtained by the classical method of ultracentrifugation/precipitation correlate well. The correlation coefficients for high-density, low-density, and very-low-density lipoproteins are 0.85, 0.99, and 0.99, respectively. Thus the densitometric method for lipoprotein analysis appears to be suitable for routine clinical application.

Improved Techniques for Assessment of Serum Lipoprotein Patterns. II. Rapid Method for Diagnosis of Type III Hyperlipoproteinemia without Ultracentrifugation

1973 ◽  
Vol 19 (10) ◽  
pp. 1139-1141 ◽  
Author(s):  
Heinrich Wieland ◽  
Dietrich Seidel
Keyword(s):  
Rapid Method ◽  
Serum Lipoprotein ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Agarose Gel ◽  
Low Density ◽  
Electrophoretic Separation ◽  
Very Low Density Lipoproteins ◽  
Type Iii ◽  
Type Iii Hyperlipoproteinemia

Abstract Based on the previously described technique [Clin. Chem.19, 737 (1973)] of precipitating plasma lipoproteins with polyanions after their electrophoretic separation in gels, a new method is presented for diagnosing type III hyperlipoproteinemia without need for ultracentrifugation or immunologic techniques. Used in the procedure are 0.1 mol/liter MgCl2, 1.5 g/liter heparin, and 10 g/liter NaCl to visualize selectively the very-low-density lipoproteins in agarose gel after electrophoresis. The technique is simple, inexpensive, accessible to every laboratory, and provides the answer in less than 2 h after electrophoresis of the patient’s whole serum. The results obtained are the same as those obtained by ultracentrifugation followed by lipoprotein electrophoresis of the isolated fractions.

scholarly journals Membrane receptors for very low density lipoprotein (VLDL) inhibitor of lymphocyte proliferation

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1055-1064 ◽  
Author(s):  
PI Yi ◽  
G Beck ◽  
S Zucker
Keyword(s):  
Dna Synthesis ◽  
Lymphocyte Proliferation ◽  
Human Lymphocytes ◽  
Low Density Lipoproteins ◽  
Membrane Receptors ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Scatchard Analysis ◽  
Low Density ◽  
Very Low Density Lipoproteins

Abstract Physiologic concentrations of human plasma very low density lipoproteins inhibit the DNA synthesis of lymphocytes stimulated by allogeneic cells or lectins. In this report we have compared the effects of isolated lipoproteins [very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)] and lipoprotein-depleted plasma (LDP) on DNA synthesis by phytohemagglutinin-stimulated human lymphocytes. The relative potency for the inhibition of lymphocyte proliferation was VLDL greater than LDL greater than HDL greater than LDP. Fifty percent inhibition of DNA synthesis was observed at a VLDL protein concentration of 1.5--2.0 microgram/ml. We have further demonstrated the presence of specific receptors for VLDL on human lymphocytes. Native VLDL was more effective than LDL in competing for 125I-VLDL binding sites. Subsequent to binding to lymphocytes, 125I-VLDL was internalized and degraded to acid- soluble products. Based on a Scatchard analysis of VLDL binding at 4 degrees C, the number of VLDL receptors per lymphocyte was estimated at 28,000 +/- 1300. Based on an estimated mean binding affinity for the VLDL receptor complex at half saturation of approximately 8.8 X 10(7) liter/mole, it is estimated that 91% of lymphocyte VLDL receptors are occupied at physiologic VLDL concentrations in blood. Although the immune regulatory role of plasma lipoproteins is uncertain, we suggest tha VLDL and LDL-In may maintain circulating blood lymphocytes in a nonproliferative state via their respective cell receptor mechanisms.

The levels of apolipoprotein-E in hypercholesteroiemic rat serum

1978 ◽  
Vol 56 (3) ◽  
pp. 161-166 ◽  
Author(s):  
Laurence Wong ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Apolipoprotein E ◽  
Fold Increase ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Rat Serum ◽  
Apo E ◽  
Control Serum ◽  
Lipoprotein Complex

The levels of apolipoprotein-E (apo-E) in serum and isolated lipoproteins from diet-induced hypercholesterolemic, and to some extent, hypertriglycerdemic rats were measured by electroimmunoassay. The hypocholesterolemia was accompanied by a mild hypertriglyceridemia. The apo-E was increased by 60% in the hypercholesterolemic serum with a 5- and 50-fold increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) respectively. However, the proportion of apo-E in nascent VLDL isolated from the hepatic Golgi apparatus of hypercholesterolemic rats was significantly decreased. In control serum, 40–50% of the apo-E is found in the density >1.21 g/ml fraction, although this is at least partially due to ultracentrifugation. The aproprotein is absent from the density >1.21 g/ml fraction from hypercholesterolemic serum, suggesting that it is bound more firmly to the lipoprotein complex. It is concluded that the large increases in apo-E in the VLDL and LDL density ranges of serum from hypercholesterolemic rats may in part be accounted for by the utilization of apo-E normally found at higher densities.

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