Rat Liver Cell Plasma Membranes Research Articles

Polymerized albumin receptor on rat liver cells.

The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the autofluorescence of rat liver plasma membranes

The autofluorescence of isolated rat liver cell plasma membranes was characterized in vitro in relation to the autofluorescence used previously for fluorescence recovery after photobleaching (FRAP) studies. The fluorescence of membrane preparations displayed an emission pattern with a maximum at around 525 nm when excited with a 468 nm blue light. The excitation spectrum monitored at 525 nm closely resembled that of flavin compounds (riboflavin, FAD, FMN). The chloroform extract of the membrane fraction showed practically no fluorescence, whereas, both the water-soluble and water-insoluble protein fractions remaining after chloroform extraction were strongly fluorescent. The fluorescence disappeared almost completely under the effect of sodium hydrosulfite, and recovered after oxidation either by shaking in air or by adding buffered hydrogen peroxide solution. The fluorescence of the acid extract of the plasma membranes photolyzed in an alkaline medium was quite similar to that of lumiflavin obtained from the photolysis of riboflavin in an alkaline medium. The plasma membranes prepared from isolated hepatocytes (which were completely devoid of endothelial cell contamination) exhibited the same autofluorescence in the liver cell plasma membranes. The results suggest that the autofluorescence of the liver cell plasma membranes is most likely of a character similar to that of flavin, bound to hepatocyte plasma membrane proteins. This fluorescence is suitable for measuring the average lateral diffusion constant of proteins by means of FRAP methods.

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Electron cytochemical and biochemical analysis of canaliculus-enriched liver cell membranes

The effects of enzyme digestion procedures were examined on rat liver cell plasma membrane preparations enriched in bile canaliculi BC). The ruthenium red-positive surface coat RRSC) was completely removed by papain and trypsin. The 50- to 60-A globular knobs on the outer surface of the BC membranes were also removed from these membranes by papain and trypsin concomitant with release of leucyl-β-naphthylamidase LAPase) activity from the membrane, suggesting that LAPase is localized on the outer luminal) surface of the BC membranes, possibly in the RRSC. Mg2+-ATPase and 5′-nucleotidase activities were found cytochemically even after papain and trypsin digestion and they were not significantly released from the membrane by these digestion procedures. However, both Mg2+-ATPase and 5′-nucleotidase activities were reduced by phospholipase C treatment and there was a decrease in the content of membrane phospholipids. Cytochemically, lack of demonstrable enzyme activities was confined to focally perturbed portions of the BC membranes. These results suggest that Mg2+-ATPase and 5′-nucleotidase are integral membrane proteins of the BC membranes, and that their activities depend upon the structural integrity of the membrane phospholipids. Pericanalicular microfilaments were firmly attached to the inner surface of the isolated BC membranes, were unaffected by phospholipase C or neuraminidase, and were minimally affected by hyaluronidase but were removed by papain and trypsin. The selective removal of both the glycoprotein-rich surface coat and the cytoskeletal components by trypsin and papain without significantly affecting the ATPase and AMPase activities is compatible with unlinkage of these peripheral membrane components. The results described with trypsin and papain are essentially the same as those found in experimental cholestasis, and raise the question whether proteases or deficiency of protease inhibitors may be more important than generally realized in the mechanism of intrahepatic cholestasis.

Biochemical and morphological changes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat liver cell plasma membrane.

The administration of single oral doses of 200 micrograms kg-1 of 2,3,7,8-tetrachlorodibenzo-p-dioxin to females of the outbred, Lac : P strain of rat results in the formation of hepatic multinucleate cells by cell fusion. Liver cell plasma membranes isolated 6 or 11 days after dosing show two distinct changes. The first is a decrease of the activity of K+-Mg2+-ATPase, which confirms histochemical observations. The second is the formation, in those animals showing a more severe intoxication, of a population of plasma membranes which are less dense than usual and which consist of extended membrane sheets. It is suggested that these alterations are consequent on a disturbance of lipid metabolism in the hepatic cells and that they represent early manifestations of the toxic process which leads to the formation of multinucleate cells.

Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane.

Uptake of bilirubin, sulfobromophthalein (BSP), and other organic anions by the liver is a process with kinetics consistent with carrier mediation. The molecular basis of this transport mechanism is unknown. In the search for the putative organic anion carrier or receptor, the interaction of BSP with rat liver cell plasma membrane (LPM) has been studied. Specific binding of [(35)S]BSP to LPM was determined using a filtration assay. Results revealed high affinity (K(a) = 0.27 muM(-1)), saturable (6.3 nmol/mg protein) binding, which was eliminated after preincubation with trypsin. Although [(35)S]BSP was strongly bound to LPM, the binding was rapidly reversible, preventing direct identification and study of a specific binding site(s). To avoid this problem, a photoaffinity probe was devised, in which [(35)S]BSP is covalently bound to LPM after exposure to ultraviolet light. Subsequent sodium dodecyl sulfate gel electrophoresis and fluorography revealed radioactivity predominantly associated with a single 55,000-mol wt protein. A protein with identical electrophoretic mobility was purified from deoxycholate solubilized LPM after affinity chromatography on glutathione-BSP-agarose gel. This protein migrated as a single band on sodium dodecyl sulfate gel electrophoresis and on urea gel isoelectric focusing. It contained 1-2 residues of sialic acid per 55,000-dalton protein, and was immunologically distinct from rat albumin and ligandin. It bound bilirubin with a K(d) of 20 muM, as determined by tryptophan fluorescence quenching. Although the high affinity of this LPM protein for organic anions suggests that it may function as a hepatocellular organic anion receptor, its role in transport of these compounds is unknown.

An organic anion binding protein isolated from rat liver cell plasma membrane (LPM)

An organic anion binding protein isolated from rat liver cell plasma membrane (LPM)

Lectin-binding sites are found in rat liver cell plasma membrane only on its extracellular surface.

In the present study ultrastructural localization of binding sites for 5 lectins was studied in rat liver cell surface membrane fractions. For this purpose ferritin-coupled Concanavalin A, wheat germ agglutinin, soybean agglutinin, Ricinus communis agglutinin 120 and Lotus tetragonolobus agglutinin I were used as probes for mannose, N-acetyl glucosamine, N-acetyl galactosamine, galactose and fucose moieties in glycoproteins and glycolipids. Although recent reports suggest presence of glycogroups on the cytoplasmic surface of cellular membranes ultrastructural identification of membrane surfaces in the present study indicated an asymmetric localization of lectin-binding sites exclusively on the extracellular side of the membranes.

Characterization of rat liver cell plasma membranes.

A method is described by which bile canalicular membranes (BCM) can be prepared, together with canaliculus-free plasma membrane (PM), both essentially free of contamination. The recovery of both fractions together was estimated to be 46%. The concentrations of total lipid, total phospholipid and cholesterol were substantially greater in the BCM, and polyacrylamide gel electrophoresis revealed differences in protein composition. The differences in lipid and protein composition of these two plasma membrane fractions are presumably related to their very different physiological functions.

A rapid high yield method for the preparation of rat liver cell plasma membranes

This report describes a simple method for the simulatenous isolation of a number of samples of rat liver plasma membranes, with high yield, purity and reproducibility. Yield and purity are discussed in terms of criteria used in other published procedures.

A simple method for the isolation of rat liver cell plasma membranes in isotonic sucrose

A method for the isolation of plasma membranes from rat liver parenchymal cells is described. It involves homogenization of parenchymal cell-enriched liver pulp in isoosmotic sucrose in a loose-fitting Dounce homogenizer. The nuclear fraction containing membranes is recovered by centrifugation. This is followed by elution of plasma and other membranous elements with either isotonic saline, isotonic saline containing 0.5 mM CaCl 2 or isotonic saline containing 2 mM EDTA and their final purification by discontinuous sucrose density gradient centrifugation. The plasma membranes are recovered at the 1.16–1.18 sucrose density interface: with isotonic saline, 0.37 mg plasma membrane protein per g wet weight rat liver is recovered. A comparison with other methods indicates that the preparation described is the simplest one available presently for cell homogenates made in isoosmotic sucrose.

Separation and partial characterization of rat liver cell membrane components.

Abstract The objectives of this research were to separate and partially characterize the components of rat liver cell plasma membranes. The plasma membranes of liver cells obtained from normal Wistar male rats were isolated using density gradients. The purified membrane was then solubilized in phenol-urea-acetic acid and gel electrophoresis was performed on the solubilized membrane components. Samples of the eluate collected following preparative gel electrophoresis were phosphorus positive-ninhydrin positive; phosphorus negative-ninhydrin positive; phosphorus po6itive-ninhydrin negative; and phosphorus negative-ninhydrin negative. The amino acid analyses showed that the proteins of the separated components were different and that no one component contained more than 6% of the total membrane protein. Liver cell plasma membranes contain components that differ from those of red blood cell plasma membranes. These components can be further divided into subgroups of proteins, i.e., phospholipoproteins and prot...