The effect of vitamin E deficiency on enzyme activity and the status of the membrane fraction of rat liver microsomes

The vitamin E deficiency was studied for its effect on the activity of enzymes participating in metabolism of xenobiotics. Experiments with 54 rats have demonstrated that the maintenance of animals on the vitamin-E-deficient diet within 13-14 weeks decreases the activity of microsomal monooxygenases (demethylase and hydroxylase), NADH- and NADPH-reductases, aryl- and aliesterases in the liver and lungs, which is a result of disturbance of hydrophobic and polar interactions in microsomal membranes. Vitamin E deficiency makes the extent of solubilization of these enzymes higher under the influence of deoxycholate and trypsin and intensifies inactivation of these enzymes under the effect of urea. In the lungs and in the liver of the vitamin E deficient rats the content of reduced glutathione decreases as well as the activity of glutathione reductase, glutathione-S-transferase, aldehyde dehydrogenase, while the activity of gamma-glutamyltransferase increases; glutathione disulphide is accumulated.     

Solubilization of the Semliki Forest virus membrane with sodium deoxycholate.

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Sodium Laurate,a Novel Protease- and Mass Spectrometry-Compatible Detergent for Mass Spectrometry-Based Membrane Proteomics

The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

ON THE SPATIAL ARRANGEMENT OF PROTEINS IN MICROSOMAL MEMBRANES FROM RAT LIVER

Rat liver rough microsomes were labeled enzymatically with ¹²⁵I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [³H]-NaBH₄ after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.     

Carbohydrates in the liver microsome fraction of rats with hypovitaminosis A

Accessibility for trypsin and sodium deoxycholate was determined in carbohydrates of glycoproteins in the liver microsome fraction of rats, which were kept for 80 days on retinol-deficient diet and received optimal amounts of vitamin A. It was found that the prevailing amount of hexose- and glucosamine-containing glycoproteins is located on the outer surface of membrane vesicles and only a smaller part of these proteins is submerged into the lipid layer of the membrane or is located on its inner surface. Above a half of protein bound with fucose and neuraminic acid is located in the lipid layer. Retinol deficiency leads to translocation of a portion of fucose- and hexose-containing proteins on the outer surface of vesicles and to a decrease of the share of these proteins in the hydrophobic membrane zone.     

Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S; effects of biological detergents

Rabbit brain cortical membranes incubated with carbachol in the presence of GTP gamma S show a marked increase in the degradation of exogenous phosphatidylinositol 4,5-bisphosphate. This activation of phospholipase C is dependent on the presence of deoxycholate and maximal at 0.8-1 mM deoxycholate. There is negligible activation by carbachol alone but in the presence of GTP gamma S a carbachol effect can be readily demonstrated. Optimal activation of phospholipase C by carbachol was seen at 10 to 100 nM free Ca2+. Washing cortical membranes with hypertonic buffer extracted 60% of the membrane protein yet the carbachol and GTP gamma S coupling remained intact. Incubation of the membranes with lysophosphatidylcholine, Nonidet P-40, sodium deoxycholate or digitonin at concentrations considerably less than those frequently used to solubilize membrane proteins abolished the carbachol response. Octyl glucoside and sodium cholate also uncoupled receptor regulation of phospholipase C but only at concentrations where solubilization of membrane proteins occurred. Prior exposure of membranes to carbachol did not prevent the uncoupling observed as a result of detergent treatment. Incubation of the membranes with carbachol and GTP gamma S did not appear to be accompanied by specific release of either active phospholipase C or inhibitors of phospholipase C activity.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.     

Isopycnic centrifugation of rat-liver microsomes in isoosmotic gradients of Percoll® and release of microsomal material by low concentrations of sodium deoxycholate

Rat-liver microsomes were subjected to isopycnic centrifugation under isoosmotic conditions in gradients of Percoll® containing 0%, 0.05%, 0.1% and 0.2% sodium deoxycholate, respectively. The buoyant density of the microsomes was in the range 1.043 to 1.046 g/ml, independent of the detergent concentration. Absorbance measurements and polyacrylamide gel electrophoresis revealed that the detergent causes an increase in the release of material from the microsomes. Electron microscopy studies showed that membrane disassembly was avoided if the microsomes were isolated at concentrations below 0.1% sodium deoxycholate.     

Structural rearrangements in membranes induced by anesthetics]

Studies were carried out on the effects of anesthetics on the stability of membranes to sodium dodecylsulfate and trypsin. The initial rate of membrane desintegration by detergent evaluated by light scattering with stopped flow method was increased in the presence of ethanol, propanol, benzyl alcohol, procain-amide (erthrocyte membrane) and chlorpromazine (rat brain synaptosomes). Concentrations of anesthetics which correspond to half of the maximum effect were very close to that of blocking the action potential and inducing 50%--antihemolysis. Alcohols and procaine at anesthetic concentrations inhibited proteolysis of isolated erythrocyte membranes but were without effect on their ultrasonic fragments. Increase of membrane desintegtation rate is interpreted as structural rearrangements of membranes with the weakening of detergent sensitive intermolecular interactions. Proteolysis inhibition of ghosts but not the gragments indicates against the participation of the basic func of proteins in the structural membrane rearrangements.     

Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest virus have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at $\text{0.9 ± 0.1}\text{mM}$ free equilibrium concentration when $\text{2.2 ± 0.2 ß 10}{}^3\text{mol}$ of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at $\text{1.5 ± 0.1}\text{mM}$ sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.     

Hepatic membrane proteins involved in ribosome binding: identification by three procedures.

Rat liver ribosomes, isolated from rough-surfaced endoplasmic reticulum using non-ionic detergent in the presence of 25 mM KCl, were associated with non-ribosomal proteins, presumably of membranous origin. These proteins could be isolated by extracting such ribosome fractions with either deoxycholate or non-ionic detergents at higher concentrations of KCl. Analysis of the extracts by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed the presence of a number of discrete polypeptides having the following approximate molecular weights: 166,000, 107,000, 100,000, 65,000 and 36,000. Ribosomes associated with the membrane-derived proteins reattached to degranulated membranes in vitro less well than did ribosomes prepared in ways which removed the proteins. Extraction of a set of similar proteins from degranulated endoplasmic reticulum by treatment with buffered 1 M urea, also interfered with ribosome reattachment. A third approach to the identification of proteins associated with ribosome attachment sites involved the labelling with radioactive succinic anhydride of apparently similar proteins in degranulated membranes, after prior treatment of the latter, before removal of bound ribosomes, with unlabelled reagent. The results indicate that certain membrane proteins may be part of the receptor sites for binding of ribosomes to the endoplasmic reticulum in rat liver.     

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components

The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23 degrees C. Some of the biological activities of the membrane survived this treatment. The leucine beta-naphthylamidase activity was more readily soluble than the 5'-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0 degrees C. Membrane solubilized in 2% deoxycholate at 23 degrees C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5'-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [(14)C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5'-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.     

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: a comparative study of spinach SoPIP2;1 and E. coli AqpZ

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.     

Vitamin A and microsomal membranes: the effect of retinol deficiency on lipid microviscosity and phospholipid turnover in rat liver microsomes

Studies with the use of the fluorescent probe pyrene revealed that vitamin A deficiency in maturing male rats results in the increased microviscosity of liver lipids. This effect seems to be due to changes in the lipid composition of microsomal membranes (increased cholesterol/phospholipid ratio and lowered polyunsaturated fatty acid content) as well as to the low level of retinol. Analysis of microsomal phospholipids labeled with [3H]palmitate and [14C]glycerol revealed that vitamin A deficiency accelerates the turnover of the glycerol skeleton but sharply decelerates that of fatty acid residues. It is concluded that the observed effect of retinol on the structural and functional properties of biological membranes is due to its ability to control the microviscosity and turnover of membrane lipids.     

Topological studies on rat liver microsomal cholesterol ester hydrolase

Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal asymmetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles.     

Dolichol and retinol content of rat liver sinusoidal cells after chronic monensin treatment.

Aim of this study was to ascertain whether an impairment of communication between parenchymal and non-parenchymal liver cells involves vitamin A intercellular transport. The following approach was adopted: liver cells were isolated from rats treated chronically with the hydrophobic ionophore monensin i.p. for 3, 5, and 7 weeks and their retinol and dolichol content was assessed. Monensin, which alters membrane flow, was used because it had previously been reported to induce liver steatosis, cholestasis and glycogenolysis after acute treatment and, by preliminary morphological examination, to impair vitamin A transport between stellate cells and hepatocytes. Dolichol was chosen as a biochemical marker because it is a membrane lipid that modulates the fluidity and permeability of the membranes that retinol must cross. After monensin treatment, a load of vitamin A was given to rats three days before sacrifice, to ascertain whether its uptake by sinusoidal liver cells was altered. The main result was a dolichol decrease in hepatocytes and in the Ito-1 subfraction. In this latter, monensin induced a decrease in dolichol content only after vitamin A load. Moreover, while the hepatocytes were able to take up a load of vitamin A normally, the Ito-1 subfraction was no longer able to store retinol. Therefore the polarised transport of retinol between hepatocytes and stellate cells seemed impaired. The behaviour of sinusoidal endothelial cells and Kupffer cells might be ascribed to the functions of these cells and is not significantly modified by monensin. In conclusion, the altered cross-talk between sinusoidal cells in liver pathology might involve retinol as well as cytokines. Different pools of dolichol might have a role in this membrane process in a hydrophobic environment.