scholarly journals B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes.

1979 ◽  
Vol 80 (1) ◽  
pp. 37-52 ◽  
Author(s):  
E D Jarasch ◽  
J Kartenbeck ◽  
G Bruder ◽  
A Fink ◽  
D J Morré ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Cytochrome B5 ◽  
High Ratio ◽  
Marker Enzyme ◽  
Microsomal Cytochrome B5 ◽  
Membrane Bound ◽  
Cytochrome P 450

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver cells

1976 ◽  
Vol 20 (1) ◽  
pp. 167-182
Author(s):  
S. Hodson ◽  
G. Brenchley
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Membrane ◽  
Rat Liver ◽  
Published Data ◽  
Protein Subunits ◽  
Electrophoretic Patterns ◽  
Close Relationship ◽  
Rat Liver Cells ◽  
Membrane Bound

A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5′-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane.

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

1988 ◽  
Vol 66 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Jon G. Church ◽  
Shobha Ghosh ◽  
Basil D. Roufogalis ◽  
Antonio Villalobo
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Line ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Normal Adult ◽  
Plasma Membrane Proteins ◽  
Phosphoprotein Phosphatase ◽  
Membrane Bound

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [γ-32P]ATP or [γ-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform–methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.

scholarly journals A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 781-788 ◽  
Author(s):  
F. Morin ◽  
S. Tay ◽  
H. Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Molecular Structures ◽  
Endoplasmic Reticulum Membrane ◽  
Marker Enzyme ◽  
Membrane Phospholipids

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.

scholarly journals Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver

1991 ◽  
Vol 280 (2) ◽  
pp. 295-302 ◽  
Author(s):  
A H Futerman ◽  
R E Pagano
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Marker Enzyme ◽  
Luminal Surface ◽  
Proteolytic Digestion ◽  
And Topology

We examined the intracellular site(s) and topology of glucosylceramide (GlcCer) synthesis in subcellular fractions from rat liver, using radioactive and fluorescent ceramide analogues as precursors, and compared these results with those obtained in our recent study of sphingomyelin (SM) synthesis in rat liver [Futerman, Stieger, Hubbard & Pagano (1990) J. Biol. Chem. 265, 8650-8657]. In contrast with SM synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis was more widely distributed, with substantial amounts of synthesis detected in a heavy (cis/medial) Golgi-apparatus subfraction, a light smooth-vesicle fraction that is almost devoid of an endoplasmic-reticulum marker enzyme (glucose-6-phosphatase), and a heavy vesicle fraction. Furthermore, no GlcCer synthesis was detected in an enriched plasma-membrane fraction after accounting for contamination by Golgi-apparatus membranes. These results suggest that a significant amount of GlcCer may be synthesized in a pre- or early Golgi-apparatus compartment. Unlike SM synthesis, which occurs at the luminal surface of the Golgi apparatus, GlcCer synthesis appeared to occur at the cytosolic surface of intracellular membranes, since (i) limited proteolytic digestion of intact Golgi-apparatus vesicles almost completely inhibited GlcCer synthesis, and (ii) the extent of UDP-glucose translocation into the Golgi apparatus was insufficient to account for the amount of GlcCer synthesis measured. These findings imply that, after its synthesis, GlcCer must undergo transbilayer movement to the luminal surface to account for the known topology of higher-order glycosphingolipids within the Golgi apparatus and plasma membrane.

scholarly journals Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver

1990 ◽  
Vol 266 (2) ◽  
pp. 363-369 ◽  
Author(s):  
M Trinchera ◽  
R Ghidoni
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Organic Phase ◽  
Time Course ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
Ganglioside Biosynthesis ◽  
The Individual ◽  
Liposomal Dispersion

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.

scholarly journals Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

1985 ◽  
Vol 101 (5) ◽  
pp. 1757-1762 ◽  
Author(s):  
N Morel ◽  
J Marsal ◽  
R Manaranche ◽  
S Lazereg ◽  
J C Mazie ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Cholinergic Nerve Terminals ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Presynaptic Plasma Membrane ◽  
Glycera Convoluta

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

scholarly journals Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

1987 ◽  
Vol 104 (5) ◽  
pp. 1239-1248 ◽  
Author(s):  
E S Sztul ◽  
D Biemesderfer ◽  
M J Caplan ◽  
M Kashgarian ◽  
J L Boyer
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Alpha Subunit ◽  
Gamma Glutamyl Transferase ◽  
Surface Distribution ◽  
Western Blots ◽  
Glutamyl Transferase

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

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