Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.     

The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes.

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of 'signet-ring' profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.     

Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C.

When isolated hepatocytes are incubated with phosphatidylinositol-specific phospholipase C, three cell-surface enzymes show markedly different behaviour. Most of the alkaline phosphatase is released at very low values of phosphatidylinositol hydrolysis, whereas further phosphatidylinositol hydrolysis releases only a maximum of about one-third of the 5'-nucleotidase. Alkaline phosphodiesterase I is not released. If cells containing phosphatidyl[3H]inositol are similarly treated, then the released [3H]inositol is in the form of inositol phosphate: no evidence has been obtained for any covalent association between released [3H]inositol and alkaline phosphatase.     

Cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer

The lateral organization of lipids and proteins in cell membranes is recognized as an important factor in several cellular processes. Cholesterol is thought to function as a modulator of the lateral segregation of lipids into cholesterol-poor and cholesterol-rich domains. We investigated how the affinity of cholesterol for different phospholipids, as seen in cholesterol partitioning between methyl-β-cyclodextrin and large unilamellar vesicles, was reflected in the lateral organization of lipids in complex bilayers. We especially wanted to determine how the low-T_m lipid affected the lateral structure. Partition experiments showed that cholesterol had a higher affinity for N-oleoyl-sphingomyelin (OSM) than for palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers, but the highest preference was for N-palmitoyl-sphingomyelin (PSM)-containing bilayers. Partial phase diagrams of POPC/PSM/cholesterol and OSM/PSM/cholesterol bilayers at 23°C and 37°C were used to gain insight into the lateral organization of lipids in bilayers. Analysis of phase diagrams revealed that the phospholipid composition of cholesterol-poor and cholesterol-rich domains reflected the affinity that cholesterol exhibited toward bilayers composed of different lipids. Therefore, the determined affinity of cholesterol for different phospholipid bilayers was useful in predicting the cholesterol-induced lateral segregation of lipids in complex bilayers.     

Transverse asymmetry of phospholipids in subcellular membranes of rat liver

Subcellular membranes isolated from rat liver in a form impermeable to macromolecules were treated with phospholipase A₂ from Naga naja venom. The phosphatidylserine, phosphatidylethanolamine and about half of the phosphatidylcholine of microsomes, Golgi membranes, inner mitochondrial membranes, lysosomes and nuclear membranes were hydrolyzed. It is proposed that these phospholipids are localized in the outer surface of the membrane bilayer, which represents the cytoplasmic side in the living cell, while the remaining phosphatidylcholine and most of the phosphatidylinositol, sphingomyelin and cardiolipin may be assigned to the inner side of the bilayer.     

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes.

The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.     

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte.

1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.     

Epinephrine-binding plasma-membrane antigens in rat liver.

Detergent extracts of isolated rat liver plasma membranes were analysed in two-dimensional immunoelectrophoresis against antiserum to plasma membranes. Enzyme staining of the immunoprecipitates revealed the presence of about ten antigens with nucleoside di- and triphosphatase activity. Most of these were earlier shown also to be NADH-neotetrazolium reductase active. In addition, two of these antigens exhibited L-leucyl-beta-naphthylamidase activity. As judged from autoradiography these plasma membrane antigens earlier characterized as multienzyme complexes bound [14C]epinephrine, and the same antigens were labelled regardless of whether membranes or membrane extracts were incubated with the radioactive hormone. The specificity of this binding was established in displacement experiments with unlabelled hormones or their analogues. Another hormone-binding antigen, also identified in the plasma membrane extract did not exhibit any known enzyme activity while three antigens with different enzyme activities had no epinephrine-binding capacity. [14C]Epinephrine-labelled plasma membrane extracts were chromatographed on Sepharose 4B and the fractions obtained were analysed in two-dimensional immunoelectrophoresis combined with autoradiography. Nucleoside di- and triphosphatases of high molecular weights (5000000) were associated with L-leucyl-beta-naphthylamidase activity, while no such associations were detected in a lower molecular weight region (70000). Further immunological studies on the various fractionated antigens provided evidence that at least two of them occurred in both low and high molecular weight fractions. Hormone-binding membrane components in varying concentrations were found throughout the eluted extract.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes

Summary The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.     

Complement-mediated production of plasma-membrane vesicles from rat fat-cells.

1. Rat isolated fat-cells were coated with rabbit anti-(rat erythrocyte) antibody and incubated with fresh guinea-pig serum for 25 min at 37 degrees C, which resulted in a more than 95% release of the cytosolic enzyme lactate dehydrogenase. 2. Under these conditions fragmentation of the plasma membrane was examined by following the plasma-membrane markers 5'-nucleotidase, adrenaline-sensitive adenylate cyclase and membrane-bound rabbit immunoglobulin G through a differential-centrifugation fractionation procedure. 3. Approx. 50% of the plasma-membrane markers remained associated with triacylglycerol. Of the remainder more than half was pelleted by centrifugation at 10 000 g for 30 min. 4. The 10 000 g supernatant was fractionated by centrifugation on a sucrose density gradient (15-50%, w/w). This procedure resulted in the production of two visible white bands on the density gradient. The bands consisted of vesicles derived from the plasma membrane, since they coincided with peaks of 5'-nucleotidase activity, contained membrane-bound immunoglobulin G and the denser one had adenylate cyclase activity. The phospholipid and protein contents of the vesicles were determined and compared with those in purified plasma membrane. 5. It is suggested that complement-mediated lysis of rat fat-cells caused the production of plasma-membrane vesicles that differ in composition from the whole plasma membrane.     

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.     

Transfer of phospholipids between the endoplasmic reticulum and mitochondria in rat hepatocytes in vivo

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling $\text{in}\text{vivo}$. With [³H]glycerol microsomal phosphatidylethanolamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occurred only after about 5 hours. It appears that the $\text{in}\text{vivo}$ transfer of these phospholipids between the two membrane compartments is a relatively slow process.     

Insulin activates the plasma-membrane and dense-vesicle cyclic AMP phosphodiesterase in hepatocytes by distinct routes.

Insulin elicits the activation of two distinct membrane-bound cyclic AMP phosphodiesterases when incubated at 37 degrees C for 5 min with intact hepatocytes: the 'dense-vesicle' enzyme and the peripheral-plasma-membrane enzyme. In hepatocytes the lysosomotropic agents chloroquine, methylamine and NH4Cl, as well as intracellular ATP depletion elicited by fructose or incubation with insulin at 22 degrees C, blocks selectively the activation of the 'dense-vesicle' enzyme. Incubation of hepatocytes with bacitracin, leupeptin and a variety of proteinase inhibitors failed to affect insulin's activation of these two cyclic AMP phosphodiesterases by distinct routes. It is suggested that activation of the 'dense-vesicle' enzyme occurs through a pathway triggered by the endocytosis, processing and recycling of the insulin receptor. This might involve the delivery, with subsequent activation, of a latent phosphodiesterase into this fraction.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

Adsorption of monovalent cations to bilayer membranes containing negative phospholipids.

The electrophoretic mobilities of multilamellar phosphatidylserine vesicles were measured in solutions containing monovalent cations, and the xi potentials, the electrostatic potentials at the hydrodynamic plane of shear, were calculated from the Helmholtz--Smoluchowski equation. In the presence of 0.1 M lithium, sodium, ammonium, potassium, rubidium, cesium, tetraethylammonium, and tetramethylammonium chloride, the xi potentials were -60, -62, -72, -73, -77, -80, -82, and -91 mV, respectively. Similar results were obtained with phosphatidylglycerol vesicles; different results were obtained with cardiolipin, phosphatidylinositol, and phosphatidic acid vesicles. The phosphatidylserine results are interpreted in terms of the Stern equation, a combination of the Gouy equation from the theory of the diffuse double layer, the Boltzmann relation, and the Langmuir adsorption isotherm. Evidence is presented that suggests the hydrodynamic plane of shear is 2 A from the surface of the membrane in solutions containing the alkali metal cations. With this assumption, the intrinsic association constants of the above monovalent cations with phosphatidylserine are 0.8, 0.6, 0.17, 0.15, 0.08, 0.05, 0.03, and 0 M-1, respectively. The validity of this approach was tested in two ways. First, the xi potentials of vesicles formed from mixtures of phosphatidylserine and a zwitterionic lipid, phosphatidylcholine, were measured in solutions containing different concentrations of sodium. All the data could be described by the Stern equation if the "relaxation" of the ionic atmosphere, which is predicted by classic electrostatic and hydrodynamic theory to occur at low salt concentrations and high potentials, was circumvented by using only large (diameter greater than 13 micrometers) vesicles for these measurements. Second, the fluorescent probe 2-(p-toluidinyl)naphthalene-6-sulfonate was used to estimate the potential at the surface of phosphatidylserine and phosphatidylglycerol vesicles sonicated in 0.1 M NaCl. Reasonable agreement with the predicted values of the surface potential was obtained.     

Fractionation of rat liver plasma-membrane regions by two-phase partitioning.

Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5'-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.     

Charge transfer across a single lipid-water interface causes ion pumping across the bilayer.

The photoformation of magnesium-porphyrin cations (P+) at a single lipid bilayer-water interface can pump lipophilic borate anions completely across the lipid bilayer and causes an actual reversal of the photovoltage. The system consists of a lipid bilayer containing magnesium octaethylporphyrin, an aqueous or interfacial electron acceptor on one side, and chloro- or fluoro-substituted tetraphenylborate in both aqueous electrolyte solutions. With 1-micros pulsed illumination, an immediate positive photovoltage is observed, which decreases on the microsecond and millisecond time scales. On the time scale of seconds, as the P+ cation concentration decays in reverse electron transfer, the voltage swings negative to a value almost equal to its initial value and finally decays with a half-time (approximately 20 s) longer than the time constant of the system (approximately 5 s). Thus, an ion gradient across the membrane is formed, trapped by the nonlinear relation between ion mobility and ion concentration. Continuous light illumination confirms that negative charge moves in the direction opposite that of the initial photoinduced electron transfer. Steady-state measurements indicate an ion pumping efficiency of approximately 30%. This simple mechanism may be a progenitor of photobiological ion pumps.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.