scholarly journals Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains.

1983 ◽  
Vol 96 (1) ◽  
pp. 217-229 ◽  
Author(s):  
A L Hubbard ◽  
D A Wall ◽  
A Ma
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Large Fraction ◽  
Membrane Preparation ◽  
Electron Microscope Autoradiography ◽  
Canalicular Membrane ◽  
Membrane Markers ◽  
Liver Plasma Membrane ◽  
Bile Canalicular Membrane ◽  
Plasma Membrane Preparation

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.

scholarly journals A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane

1976 ◽  
Vol 154 (1) ◽  
pp. 11-21 ◽  
Author(s):  
J P Luzio ◽  
A C Newby ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Fat Cells ◽  
Fat Cell ◽  
Cell Plasma ◽  
Rat Fat Cells ◽  
Plasma Membrane Preparation ◽  
Isolated Rat

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.

Adenosine transport in rat liver plasma membrane vesicles

1991 ◽  
Vol 261 (5) ◽  
pp. G716-G722 ◽  
Author(s):  
R. H. Moseley ◽  
S. Jarose ◽  
P. Permoad
Keyword(s):  
Plasma Membrane ◽  
Transport Process ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Plasma Membrane Vesicles ◽  
Canalicular Membrane ◽  
Liver Plasma Membrane ◽  
Adenosine Uptake ◽  
Bile Canalicular Membrane ◽  
Adenosine Transport

Liver plasma membrane ecto-ATPase activity is largely restricted to the bile canalicular membrane. To determine whether a transport process is also selectively present on this membrane surface to reclaim adenosine derived from the intracanalicular degradation of ATP, the characteristics of hepatic nucleoside transport were examined in canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles. In the presence of the adenosine deaminase inhibitor, deoxycoformycin, an inwardly directed Na+ gradient markedly stimulated [3H]adenosine uptake in cLPM vesicles. Canalicular Na(+)-dependent [3H]adenosine uptake was enhanced by an intravesicular-negative membrane potential and inhibited by dissipation of the Na+ gradient with gramicidin D. Both purine and pyrimidine nucleosides inhibited canalicular adenosine transport. 6-[(4-Nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, an inhibitor of nucleoside transport in erythrocytes and nonepithelial cells, had no effect on canalicular adenosine transport. Canalicular Na(+)-dependent [3H]adenosine uptake exhibited saturability with a Michaelis-Menten constant of 8.3 microM and a maximum transport rate of 7.6 pmol.5 s-1.mg protein-1. In contrast, [3H]adenosine uptake in blLPM vesicles was not stimulated by an inwardly directed Na+ gradient. These findings demonstrate asymmetric distribution of hepatic Na(+)-dependent nucleoside transport. Reclamation of intracanalicular adenosine resulting from ecto-ATPase activity may explain the presence of this transport process selectively on the bile canalicular membrane.

An improved procedure for the purification of plasma membranes from Dictyostelium discoideum

1977 ◽  
Vol 55 (12) ◽  
pp. 1233-1236 ◽  
Author(s):  
N. R. Gilkes ◽  
G. Weeks
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Preparation Procedure ◽  
Contamination Level ◽  
Marker Enzymes ◽  
Plasma Membrane Preparation

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. &Weeks, G. (1977) Biochim. Biophys. Acta 464, 142–156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.

Liver Cell Plasma Membrane Lipids and the Origin of Biliary Phospholipid

1975 ◽  
Vol 53 (9) ◽  
pp. 989-997 ◽  
Author(s):  
I. M. Yousef ◽  
D. L. Bloxam ◽  
M. J. Phillips ◽  
M. M. Fisher
Keyword(s):  
Plasma Membrane ◽  
Liver Cell ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Phospholipid Composition ◽  
Bile Canaliculi ◽  
Canalicular Membrane ◽  
Cell Plasma ◽  
Bile Canalicular Membrane

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with their contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phospholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes did not show the biphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over at different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remaining plasma membrane.

Steroid sequestration within the rat adrenal zona glomerulosa

1988 ◽  
Vol 117 (2) ◽  
pp. 191-NP ◽  
Author(s):  
S. M. Laird ◽  
G. P. Vinson ◽  
B. J. Whitehouse
Keyword(s):  
Plasma Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Zona Glomerulosa ◽  
Membrane Preparation ◽  
The Media ◽  
Considerable Concentration ◽  
Highly Correlated ◽  
Plasma Membrane Preparation

ABSTRACT Accumulated data from in-vitro experiments have suggested that 18-hydroxysteroids may be stored within the intact rat adrenal zona glomerulosa. The phenomenon was further investigated by comparing the amount of steroid remaining in the zona glomerulosa tissue with that secreted into the media during incubation in vitro. The results showed that 18-hydroxydeoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) were retained within the tissue against a considerable concentration gradient, with smaller amounts of aldosterone and corticosterone. Lysis of the intact zona glomerulosa, by preincubation in distilled water, yielded an enriched plasma membrane preparation. After subsequent incubation in Krebs–Ringer bicarbonate this preparation contained significantly more 18-OH-DOC than did the intact tissue, suggesting that tissuesequestered 18-OH-DOC is normally metabolized to other products. These may include 18-OH-B and aldosterone. Fractionation of homogenized intact zona glomerulosa and the enriched plasma membrane preparation by density gradient centrifugation showed that tissue 18-OH-DOC banded in fractions of density 1·063– 1·21 g/ml and that its distribution was highly correlated with protein. Corticosterone, 18-OH-B and aldosterone banded like added free [3H]18-OH-DOC in fractions of density < 1·006 g/ml. The results suggest that 18-OH-DOC is the major sequestered steroid within the rat adrenal zona glomerulosa and that this sequestration is attributable to the association of 18-OH-DOC with a high-density component of the plasma membrane. J. Endocr. (1988) 117, 191–196

scholarly journals Role of membranes in bile formation. Comparison of the composition of bile and a liver bile-canalicular plasma-membrane subfraction

1976 ◽  
Vol 154 (3) ◽  
pp. 589-595 ◽  
Author(s):  
W H. Evans ◽  
T Kremmmer ◽  
J G. Culvenor
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Bile Acids ◽  
Secretory Process ◽  
Major Protein ◽  
Outer Face ◽  
Ph Optimum ◽  
Bile Formation ◽  
Canalicular Membrane ◽  
Bile Canalicular Membrane

1. Enzymes, proteins, glycoproteins and lipids of rodent bile were compared with those of a plasma-membrane subfraction originating from the hepatocyte bile-canalicular membrane. 2. Three bile-canalicular glycoprotein enzyme activities were detected in bile. Comparison of the pH optimum and immunoinhibition properties of membrane and bile 5′-nucleotidase activity indicated that they were the same enzyme. Correspondence between membrane and bile alkaline phosphodiesterases also suggested that they were the same enzymes. Activities of Mg2+-stimulated adenosine triphosphatase, a lipid-dependent intrinsic membrane protein, and galactosyltransferase, a Golgi membrane marker, were not detected in bile. 3. Rodent bile contained 15 polypeptide bands that differed radically from those of bile-canalicular membranes. Bands that may correspond in molecular weight to liver plasma-membrane glycoproteins were present at low staining intensities in bile. A major protein of apparent molecular weight 49 500 was present, and albumin was detected by immunodiffusion. 4. The lipid composition of bile and bile-canalicular membrane also differed. Phosphatidylcholine accounted for 82% of rat bile phospholipids, and only trace amounts of phosphatidylinositol, phosphatidylserine and sphingomyelin were present. 5. The results indicate that in healthy animals, the bile-canalicular membrane is refractory to the action of bile acids during the secretory process. The presence of only small amounts of bile-canalicular membrane components, especially glycoprotein enzymes located at the outer face of the membrane, suggests that these are released from the membrane by bile acids after secretion of bile into the canalicular spaces.

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