Rat Liver Plasma Membrane Fraction Research Articles

Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver.

We isolated and characterized a subcellular fraction derived from the blood-sinusoidal plasma membrane of hepatocytes enriched in caveolin and containing several of the molecular components described to be present in caveolae isolated from other cell types. A morphological study by electron microscopy revealed that it was composed of caveolae-attached membrane profiles. Immunoelectron microscopy of isolated fraction showed the specific labeling of internal caveolae membranes with anti-caveolin antibody. Finally, one- and two-dimensional electrophoresis and Western blotting were used for the biochemical analysis of this new rat liver plasma membrane fraction. From the biochemical and the morphological characterization, we conclude that the caveolae-enriched plasma membrane fraction is a plasma membrane fraction, which originates from specialized regions of the sinusoidal plasma membrane, enriched in caveolae.

Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes.

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane.

A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R.N., and Hemer, M.R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) J. Biol. Chem. 264, 14354-14360). We now demonstrate that the addition of both Cer-1-phosphate (Cer-1-P) and a short-acyl chain analog of Cer-1-P,N-hexanoylsphingosine-1-phosphate (C6-Cer-1-P) to cultured cells and a variety of subcellular fractions results in rapid degradation to Cer and C6-Cer, respectively. The Cer-1-P phosphatase activity is enriched in a rat liver plasma membrane fraction and appears to be distinct from the phosphatase that hydrolyzes phosphatidic acid (PA), PA phosphohydrolase, as shown by the difference in sensitivity of Cer-1-P and PA hydrolysis to propranolol, detergent, and heat treatment. Moreover, the Km of Cer-1-P hydrolysis is 10-fold lower than the Km of PA hydrolysis in plasma membrane. PA is a noncompetitive inhibitor of Cer-1-P hydrolysis, with an inhibition constant 1-1.5-fold higher than the Km of Cer-1-P hydrolysis. In contrast, Cer-1-P does not inhibit PA hydrolysis. Finally, we describe the synthesis of a novel analog of Cer-1-P which is not hydrolyzed in vitro and in vivo and is internalized in cultured cells by endocytosis. These results are discussed in relation to the possible roles of Cer-1-P in regulating intracellular levels of Cer.

Iodination and binding characteristics of 125I-d(CH2)5 [Tyr(Me)2, Tyr(NH2)9] AVP

With iodinated vasopressin analogue, d(CH2)5[Tyr(Me)2, Tyr(NH2)9] AVP, at position 9, followed by purification by HPLC (specific activity 473-543Ci/mmol), a specific binding was observed in the rat liver plasma membrane fraction. Scatchard analysis indicated a single class of high-affinity binding sites with a Kd of 0.23nM and Bmax of 142fmol/mg protein. V2-agonist, DDAVP, did not displace 125I-vasopressin analogue. These results suggest that 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9] AVP with a high specific activity is a useful tool to investigate V1-receptors.

Isolation and characterization of canalicular and basolateral plasma membrane fractions from human liver

A method is described for the isolation of subfractions from human liver plasma membranes, enriched in canalicular domains (cLPM) and basolateral domains (blLPM), respectively, and the results are compared to those obtained with rat liver. The studies were performed in 18 human livers. The cLPM (isolated at densities 1.103–1.127 for human and 1.036–1.127 for rat cLPM) from human as well as rat liver showed a lower density than the blLPM (1.141–1.161 for human and 1.151–1.172 for rat biLPM). Human and rat blLPM were characterized by increased levels of (Na +/K +)-ATPase (relative enrichment 33 and 21, respectively). Both human and rat cLPM showed high specific activities of leucine aminopeptidase; relative enrichment factors were 42 and 31, respectively. Mg 2+-ATPase and alkaline phosphatase, specific canalicular enzymes in rat liver, were only sligtly enriched in the cLPM of human liver, which indicates that these enzymes are not suitable as marker enzymes for human liver cLPM. Both cLPM and bILPM of human and rat origin were only slightly contaminated with mitochondria, lyposomes, Golgi membranes and endoplasmic reticulum. Total recoveries of cLPM and blLPM were 0.02 mg protein/g liver each for the human membrane preparations, compared to 0.07 and 0.16 mg protein/g liver for the membranes prepared from rat liver. Analysis of membrane fluidity revealed that the human liver cLPM were more rigid than blLPM (mean difference in fluorescence polarization P DPH 0.024). They contained more cholesterol (0.43 vs. 0.30 μmol/mg protein) and phospholipids (0.54 vs. 0.39 μmol/mg protein, respectively), which was compatible to rat liver plasma membrane fractions. This study shows that besides similarities, there are several differences between human and rat liver plasma membrane fractions.

Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60c-yes.

A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg.ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60c-yes undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the Km for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the Vmax of copolymer and tubulin phosphorylation.

Ca 2+ transport by rat liver plasma membranes: the transporter and the previously reported Ca 2+-ATPase are different enzymes

A rat liver plasma membrane fraction showed an ATP-dependent uptake of Ca 2+ which was released by the ionophore A23187. This activity represents a plasma membrane component and is not due to microsomal contamination. The Ca 2+ transport displayed several properties which were different from those of the high-affinity Ca 2+-ATPase previously observed in these membranes (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209–11215; Birch-Machin, M.A. and Dawson, A.P. (1986) Biochim. Biophys. Acta 855, 277–285). These observations have shown that Ca 2+-ATPase does not require added Mg 2+ whereas we have demonstrated that, in the same membrane preparation, Ca 2+ uptake required millimolar concentrations of added Mg 2+. The Ca 2+-ATPase activity, with apparent K m values of 0.25 μM Ca 2+, 0.15 mM MgATP and 1.0 μM Ca 2+, 4 μM MgATP respectively. The apparent maximum rate of Ca 2+ uptake was about 150-fold less than Ca 2+-ATPase activity. These features suggest that the high-affinity Ca 2+-ATPase is not the enzymic expression of the ATP-dependent Ca 2+ transport mechanism.

Lithocholic acid-cholesterol interactions in rat liver plasma membrane fractions

This study was designed to elucidate the steps involved in the incorporation of lithocholic acid and the increase in cholesterol in liver plasma membranes after lithocholic acid injection. In vitro, cholesterol incorporation or binding to liver plasma membrane fractions enriched in bile canalicular structures occurred only when cholesterol was added simultaneously with lithocholic acid. The addition of cholic acid did not prevent the incorporation or binding of lithocholic acid and of cholesterol. However, when cholic acid was incubated with membranes already containing lithocholic acid and cholesterol, the ratio of cholesterol to lithocholic acid increased from 2 to more than 3 via a reduction of lithocholic acid. The binding of lithocholic acid and cholesterol to membranes rose 5-fold in the presence of cytosolic proteins. By electron microscopy the canalicular membrane structures with a high cholesterol content exhibited few microvilli, and their lumen appeared to have collapsed. These data suggest that simultaneous interaction of lithocholic acid and cholesterol, and not prior incorporation or binding of lithocholic acid to the membrane, may be a prerequisite to cholesterol accumulation in the membrane.

Phosphorylation of a 16-kDa protein by diacylglycerol-activated protein kinase C in vitro and by vasopressin in intact hepatocytes

A 16-kDa protein present in a purified rat liver plasma membrane fraction and also in cytosol can be phosphorylated by endogenous diacylglycerol-activated protein kinase C. In intact hepatocytes prelabeled with 32P, vasopressin causes a rapid increase in the phosphorylation of a 16-kDa protein having a similar p I value to that observed in in vitro studies. These findings suggest that vasopressin-induced phosphorylation of the 16-kDa in the intact hepatocyte may reflect increased activity of protein kinase C, secondary to membrane polyphosphoinositide breakdown. Phosphorylation of the 16-kDa protein may thus be part of the coordinated mechanism associated with hormonal regulation of cellular Ca 2+ fluxes.

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique, enzymatic profile, composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg 2+ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg 2+-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na +,K +)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na +,K +)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ( Na + + K +)- ATPase specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[ 3H]glucose into an osmotically sensitive space bounded by these membrane vesicles.

Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro

As part of a study of the mechanism whereby organic anionic dyes such as sulfobromophthalein and bilirubin enter hepatocytes, the binding of [ 35S]sulfobromophthalein and of its glutathione conjugate to two rat liver plasma membrane fractions were studied in vitro. Both fractions reversibly and saturably bound conjugated and unconjugated sulfobromophthalein. Three classes of binding site were necessary to account for the observed sulfobromophthalein binding, their maximal binding capacities being 3.5 · 10 −11, 1.6 · 10 −7 and 5.4 · 10 −7 mol/mg membrane protein. The corresponding association constants were 5.5 · 10 7, 1.5 · 10 5 and 1.3 · 10 3 M −1. Binding of the glutathione conjugate could be accounted for by two classes of binding site only, their association constants being 2.0 · 10 8 and 1.9 · 10 3 M −1 and their maximal binding capacities 5.0 · 10 −11 and 2.2 · 10 −7 mol/mg protein, respectively. Conjugated and unconjugated sulfobromophthalein mutually competed for binding, K I being 7.8 · 10 −7 and 5.5 · 10 −5 M for conjugated and unconjugated sulfobromophthalein, respectively. Similarly, bilirubin and indocyanine green, but not taurocholate, competitively inhibited sulfobromophthalein binding. Treatment with trypsin and phospholipases reduced, while treatment with neuraminidase did not affect binding. Neither changes in pH nor substitution of other cations for Na + in the incubation mixture significantly affected sulfobromophthalein binding. Heating the membranes increased binding. This was due to an increase in maximal binding capacity of the low-affinity, high-capacity sites. The description of saturable binding sites on hepatocellular surface membranes, the affinity of one of the sites exceeding the reported affinities for albumin and ligandin, supports the hypothesis that a membrane-located membrane carrier is responsible for hepatic uptake and biliary excretion of organic anionic dyes. Based on the studies with enzyme treatments, it is speculated that this carrier is a phospholipid-dependent, integral membrane protein.

Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography

As part of a study of hepatic organic anion transport, solubilized liver plasma membrane proteins were subjected to affinity chromatography on bilirubin- and sulfobromophthalein-labeled agarose columns. Both columns retained a Sudan Black and PAS negative protein of molecular weight 60,000 daltons, which cochromatographed with [ 35S]sulfobromophthalein on Sephadex G-75, and reversibly bound [ 35S]sulfobromophthalein in vitro with high affinity (K a ⋍ 10 7 M −1) and a valence of 2. Erythrocyte ghost membranes did not contain this protein. Sulfobromophthalein-agarose retained two additional smaller proteins which did not cochromatograph with [ 35S]sulfobromophthalein. Their significance is unclear. This study supports the hypothesis that liver cell plasma membranes participate in the hepatic transport of organic anions.