Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins.

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase [( 3H]galBSA-HRP), injected 10 min before sacrifice. These organelles were recovered at low density (1.11-1.13 g/ml) in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H2O2 and equilibration in a second sucrose gradient, galBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J., J. Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB-stained structures represented approximately 85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities. These minor activities could reflect either contamination or true enzyme association to the ligand-containing structures. Considering the latter hypothesis, at most 1.0% of alkaline phosphodiesterase I and 2.6% of 5'-nucleotidase (markers for plasma membrane), 3.6% of N-acetyl-beta-glucosaminidase (lysosomes), and 6.0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand-containing organelles. After DAB cytochemistry on liver fixed 10 min after galBSA-HRP injection, ligand-containing structures accounted for 0.78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2/cm3, compared with 6,700 cm2/cm3 for the pericellular membrane. Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex.

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Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Biochemical Journal ◽

10.1042/bj2320071 ◽

1985 ◽

Vol 232 (1) ◽

pp. 71-78 ◽

Cited By ~ 15

Author(s):

J A Hedo ◽

I A Simpson

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Insulin Receptor ◽

Golgi Complex ◽

Microsomal Fraction ◽

Primary Cultures ◽

Sodium Dodecyl ◽

High Density ◽

Low Density ◽

Adipose Cells

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

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CHARACTERIZATION OF RAT LIVER SUBCELLULAR MEMBRANES

The Journal of Cell Biology ◽

10.1083/jcb.60.2.460 ◽

1974 ◽

Vol 60 (2) ◽

pp. 460-472 ◽

Cited By ~ 10

Author(s):

David H. DeHeer ◽

Merle S. Olson ◽

R. Neal Pinckard

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Nuclear Membrane ◽

Membrane Fraction ◽

Cross Reactivity ◽

Selective Absorption ◽

Mitochondrial Membranes ◽

Subcellular Membrane ◽

Hepatocellular Necrosis

The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes.

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Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

Journal of Virology ◽

10.1128/jvi.76.14.6919-6928.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 6919-6928 ◽

Cited By ~ 461

Author(s):

P. André ◽

F. Komurian-Pradel ◽

S. Deforges ◽

M. Perret ◽

J. L. Berland ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Apolipoprotein B ◽

Core Protein ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Hcv Rna ◽

Low Density ◽

Density Fractions

ABSTRACT The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

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Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles

Biochemical Journal ◽

10.1042/bj2560117 ◽

1988 ◽

Vol 256 (1) ◽

pp. 117-124 ◽

Cited By ~ 25

Author(s):

C Dargemont ◽

M Hilly ◽

M Claret ◽

J P Mauger

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Uptake Rate ◽

Ionic Composition ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Microsomal Preparation ◽

Intact Cells ◽

Inside Out

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

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Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver

Biochemical Journal ◽

10.1042/bj2370163 ◽

1986 ◽

Vol 237 (1) ◽

pp. 163-173 ◽

Cited By ~ 42

Author(s):

E H Morgan ◽

G D Smith ◽

T J Peters

Keyword(s):

Rat Liver ◽

Specific Binding ◽

Bimodal Distribution ◽

Subcellular Fractionation ◽

Low Density ◽

Extracellular Medium ◽

Density Peak ◽

Electron Microscope Autoradiography ◽

Density Fractions

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 × 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.

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Reconstitution and characterization of ATP-dependent bile acid transport in human and rat placenta

Biochemical Journal ◽

10.1042/bj3110479 ◽

1995 ◽

Vol 311 (2) ◽

pp. 479-485 ◽

Cited By ~ 23

Author(s):

P Bravo ◽

J J G Marin ◽

M J Beveridge ◽

D A Novak

Keyword(s):

Plasma Membrane ◽

Bile Acid ◽

Rat Liver ◽

Brush Border ◽

Atp Hydrolysis ◽

Structural Characteristics ◽

Acid Transport ◽

Transport Activity ◽

Canalicular Membrane

Bile acid (BA) transport across the human microvillus maternal-facing trophoblast plasma membrane (mTPM) has been recently reported to be stimulated by the presence of ATP [Marin, Bravo, El-Mir and Serrano (1993) J. Hepatol. 18, S41]. Reconstitution of BA transport activity in proteoliposomes from human mTPM is reported in this paper. Typical characteristics of BA transport in native mTPM vesicles, including a requirement for ATP hydrolysis and inhibition by other BA species, were preserved in proteoliposome preparations. BA transport into 20- and 14-day-gestation rat mTPM vesicles was also stimulated by the presence of ATP as noted in human mTPM and in the rat liver canalicular membrane. Besides this functional similarity, these ATP-dependent carriers may share structural characteristics, as demonstrated by studies using an antibody (100 Ab) raised against the 100 kDa BA carrier of the canalicular membrane from rat liver which recognized proteins in both human and rat brush-border trophoblast membranes.

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Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver

Biochemical Journal ◽

10.1042/bj2520437 ◽

1988 ◽

Vol 252 (2) ◽

pp. 437-445 ◽

Cited By ~ 12

Author(s):

J M Graham ◽

D J Winterbourne

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Heparan Sulphate ◽

Membrane Vesicles ◽

Membrane System ◽

Glycoprotein Synthesis ◽

Golgi Membranes ◽

Two Populations

We report on the incorporation of radiolabelled sulphate into proteoglycan in the ‘in situ’-perfused rat liver. After 5 min virtually all of the [35S]sulphate was incorporated into heparan sulphate; no partially sulphated precursors were detected. Pulse-chase experiments, followed by centrifugation in gradients of sucrose and metrizamide, showed that, at 5 min, the heparan sulphate was associated predominantly with the Golgi membranes. Over the next 20 min, intact proteoglycan appeared at the plasma membrane. At intermediate times the heparan sulphate was detected simultaneously in two distinct populations of membrane vesicles. Whether the heparan sulphate in these two populations has two different destinies (e.g. plasma membrane or secretion) is not yet clear. Subfractionation of the Golgi membranes showed that the N-sulphotransferase co-purified with the heparan [35S]sulphate and was separable from the galactosyltransferase of glycoprotein synthesis, confirming that the Golgi membrane system is functionally segregated. Subfractionation also permitted an almost 100-fold purification of the N-sulphotransferase over the homogenate: this will provide an excellent starting material for isolation and further characterization of the enzyme.

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