Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.     

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.     

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

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.     

S-(4-azidophenacyl)[35S]glutathione photoaffinity labeling of rat liver plasma membrane-associated proteins

A method for the synthesis of the glutathione conjugate S-(4-azidophenacyl)[35S]glutathione is described. The compound was used for photoaffinity labeling of proteins present in canalicular membrane vesicles (CMV), sinusoidal membrane vesicles (SMV), mitochondria and microsomes from rat liver. Most of the radioactivity introduced by photoaffinity labeling of CMV appeared in the 25-29 kDa range. Further labeled proteins were observed in bands at 37, 105 and about 120 kDa. 79% of the 25-29 kDa associated radioactivity was recovered in the supernatant after extensive revesiculation (washing) of the vesicles, together with the 37 kDa protein. CMV and SMV contained glutathione S-transferase (GST) activity which in CMV was decreased by 75% by washing. Photolabeling of a mixture of purified basic GST subunits from rat liver resulted in a band pattern at 25-29 kDa similar to that in the membrane preparations. Isoelectric focusing of the CMV indicated the presence of basic soluble GST subunits. S-Hexylglutathione-Sepharose affinity chromatography showed reversible binding of photolabeled proteins at 25-29 kDa. Difference photoaffinity labeling with GSSG, S-hexylglutathione, taurocholate and phenylmethylsulfonyl fluoride decreased the radioactivity bound by GST, but not that introduced into the 105 kDa protein band present in CMV. It is concluded that membrane-associated basic GST isoenzymes are present in standard membrane vesicle preparations. In the cell, the function may be transport of GST-bound compounds across the membrane and protection of the membranes against electrophiles.     

Proteolytic processing of rat liver membrane secretory component. Cleavage activity is localized to bile canalicular membranes

Membrane secretory component (mSC) mediates the transcellular movement of polymeric IgA from the sinusoidal to the bile canalicular surface of rat hepatocytes. Prior to or concomitant with arrival at the bile canalicular membrane, mSC is cleaved, producing a soluble proteolytic fragment (fSC) which is released into the bile. Conversion of mSC to fSC occurs at the cell surface of cultured rat hepatocytes (Musil, L. S., and Baenziger, J. U. (1987) J. Cell Biol. 104, 1725-1733), suggesting that vectorial release of fSC into bile in vivo may reflect localization of a mSC-specific protease to bile canalicular membranes. We have established a reconstituted system to examine the process of specific cleavage of mSC to yield fSC and to characterize the protease activity responsible. A membrane fraction highly enriched for endocytic vesicles was found to contain approximately 90% of the [35S]Cys-mSC from metabolically labeled rat liver slices but only 5% of the cellular protein. No cleavage activity was present in these vesicles. Highly enriched bile canalicular membranes were able to mediate cleavage of metabolically labeled mSC to a fragment indistinguishable from authentic fSC. In the absence of nonionic detergent, cleavage was dependent on the presence of polyethylene glycol, presumably to mediate fusion of mSC-enriched membranes with bile canalicular membranes. Following solubilization with nonionic detergent, cleavage was no longer dependent on the addition of polyethylene glycol. Cleavage of mSC was not observed with either intact or detergent-solubilized sinusoidal, microsomal, or lysosomal membranes. We have thus identified a proteolytic activity associated with bile canalicular membranes which has the properties of a membrane protein and is likely to be responsible for production of fSC in vivo. Its highly restricted localization to the bile canalicular membrane would account for the vectorial release of fSC into the bile.     

Epidermal growth factor receptor in chronic bile duct obstructed rats: implications for maintenance of hepatocellular mass.

Changes of the number and properties of the epidermal growth factor (EGF) receptor occur during liver regeneration and may be of importance in the maintenance of hepatocellular mass in liver cirrhosis. We therefore studied the changes in the number and distribution of EGF receptor in the development of liver cirrhosis induced by bile duct ligation. Receptor binding assays demonstrated a marked decrease in the binding capacity of crude plasma membrane fractions from 45 +/- SD 16 to 19 +/- 10 fmol/mg protein (p < 0.001) in control and bile duct ligated livers, respectively while the Kd increased after 3 days of bile duct ligation from 0.5 +/- 0.2 to 1.4 +/- 0.6 nmol/l. Total receptor concentration in the same membrane fractions, as assessed by Western blot analysis, was not changed. The expression of EGF receptor mRNA was reduced to about one third of control levels after 28 days of bile obstruction. Immunohistochemistry, performed using monoclonal antibodies against EGF receptor, showed a strong labeling of cytoplasm (87 +/- 3% positive) and plasma membranes (84 +/- 24%) but no labeling of nuclei in control livers. In bile duct ligated rats, in contrast, cytoplasmic staining was decreased (15 +/- 12%) already after 3 days of bile obstruction; labeling of canalicular membranes and nuclei appeared after 14 days. The shift of EGF receptor from plasma membranes to nuclei supports the notion that EGF receptor is involved in the maintenance of hepatocellular mass in this model of liver cirrhosis. This concept is supported by the finding of decreased mRNA for EGF receptor presumably representing down-regulation as seen in regenerating rat liver.     

Isolation and characterization of the putative canalicular bile salt transport system of rat liver

Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.     

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive subcanalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per micron of plasma membrane were 7.86 vs 2.46 (P less than 0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P less than 0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.     

Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system

Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies.

We have produced antibodies specific for the three P-glycoprotein (P-gp) isoforms encoded by the mouse mdr1, mdr2, and mdr3 genes. The anti-Mdr2 and anti-Mdr3 antibodies were generated against synthetic peptides derived from the "linker" region, whereas the anti-Mdr1 antibody was raised against a fusion protein containing the amino terminus of Mdr1. Western blot analysis showed that the three antibodies could discriminate between the three isoforms in membrane fractions from Hamster cells transfected with the corresponding full-length or chimeric mdr cDNAs. Immunocytochemistry studies of mdr-transfected cells showed that the three antibodies specifically recognized each P-gp isoform expressed in whole cells. Immunoblotting of normal mouse tissues revealed that the Mdr2 isoform was expressed at very high levels in liver canalicular membrane vesicles (CMV) but not in membrane vesicles prepared from the basolateral (sinusoidal) domain (SMV). Mdr3 was detected in intestinal brush border membrane vesicles and also in CMV, although at levels much lower than Mdr2. Mdr1 was not detected in CMV or SMV but was detected in endometrial tissue from the gravid uterus. Photolabeling experiments with [125I]iodoarylazidoprazosin followed by immunoprecipitation with isoform-specific antibodies indicated that, in CMV, Mdr3 but not Mdr2 could bind the drug analogue.     

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.     

Preservation of hepatocyte plasma membrane domains during cell division in situ in regenerating rat liver

We have utilized antibodies against five domain-specific integral proteins of the rat hepatocyte plasma membrane to examine the fates of the plasma membrane domains during hepatocyte division in the regenerating rat liver. The proteins were quantified on immunoblots of liver homogenates prepared during the peak of hepatocyte mitotic activity, 28-30 hr after two-thirds hepatectomy. Two sinusoidal/lateral proteins, CE 9 and the asialoglycoprotein receptor, and one bile canalicular protein, dipeptidylpeptidase IV, were not changed significantly in amount; whereas one sinusoidal/lateral protein, the epidermal growth factor receptor, and one bile canalicular protein, HA 4, were reduced to less than or equal to 50% of control levels. Light microscopic examination of plastic sections of regenerating liver tissue revealed that the mitotic hepatocytes generally appeared to retain normal contacts with neighboring interphase hepatocytes. Immunofluorescence was used to localize the domain-specific proteins on mitotic hepatocytes identified in 0.5-micron frozen sections of 28- to 30-hr regenerating liver tissue. Independent of mitotic stage, the hepatocytes retained mutually exclusive bile canalicular and sinusoidal/lateral domains, as defined at the molecular level by the distributions of specific proteins, such as HA 4 and CE 9, respectively.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Summary Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive sub-canalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per m of plasma membrane were 7.86 vs 2.46 (P<0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P<0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.This study was supported by the Swiss National Science Foundation grants 3.846.0.87 (to L.L.) and 3.992.0.87 (to P.J.M.)     

CMP-N-acetylneuraminic acid hydrolase, an ectoenzyme distributed unevenly over the hepatocyte surface.

The regional localization of CMP-N-acetylneuramic acid hydrolase at the hepatocyte surface was studied by using plasma membranes and hepatocytes isolated from rat liver. 1. By homogenization of the rat liver plasma membrane preparations and subsequent discontinuous sucrose gradient centrifugation, one light and two heavy membrane fractions were obtained. The origin of these three subfractions is discussed based on the specific activities in the three fractions of 5'-nucleotidase, alakaline phosphatase and Mg2+-ATPase and on electron microscopic examination of the fractions. Evidence is given suggesting that the light fraction is derived from the bile canalicular surface of the plasma membrane, and that the heavy fractions are derived predominantly from the sinusoidal and lateral surfaces of the liver cell membrane. CMP-AcNeu hydrolase was present at highest specific activity in one of the heavy subfractions. Therefore it is concluded that CMP-AcNeu hdyrolase is located preferentially in the sinusoidal and/or lateral plasma membrane parts of the liver cell. 2. Experiments with intact and disintegrated hepatocytes isolated from rat liver indicated that CMP-AcNeu hydrolase is located at the surface of the cell membrane, with its functional group directed to the outside.     

A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain

Indirect immunofluorescence was used to establish a domain-specific marker for hepatocyte plasma membranes. In frozen sections of fixed rat liver (0.5-4 microns), antibodies directed against rat intestinal leucine aminopeptidase (LAP) recognized an antigen that was restricted to the bile canalicular plasma membrane. Fluorescence was not observed on the sinusoidal or lateral membranes, and intracellular staining was not detected. The liver antigen was identified as LAP, based on its chemical similarity to intestinal LAP. First, immunoprecipitation experiments using trypsin-solubilized intestinal LAP (G-200 fraction, 91% pure) established a correlation between the loss of LAP enzyme activity from the soluble fraction and the appearance in the specific immunoprecipitates of polypeptides migrating on SDS PAGE between 110,000 and 130,000 daltons. The antigen precipitated from a detergent extract of liver plasma membranes had the same electrophoretic mobility. Second, the chymotryptic map of the major band in the liver immunoprecipitate was similar to that of purified intestinal LAP.     

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

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 thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the 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 phosholipids, 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 diphasic 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 a 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 remainong plasma membrane.