Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase [( 3H]galBSA-HRP), injected 10 min before sacrifice. These organelles were recovered at low density (1.11-1.13 g/ml) in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H2O2 and equilibration in a second sucrose gradient, galBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J., J. Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB- stained structures represented approximately 85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities. These minor activities could reflect either contamination or true enzyme association to the ligand- containing structures. Considering the latter hypothesis, at most 1.0% of alkaline phosphodiesterase I and 2.6% of 5'-nucleotidase (markers for plasma membrane), 3.6% of N-acetyl-beta-glucosaminidase (lysosomes), and 6.0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand- containing organelles. After DAB cytochemistry on liver fixed 10 min after galBSA-HRP injection, ligand-containing structures accounted for 0.78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2/cm3, compared with 6,700 cm2/cm3 for the pericellular membrane. Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex.     

Dual subcellular localization of sialidase in cultured granule cells differentiated in culture.

A rapid small-scale procedure was set up to obtain highly purified preparations of lysosomes and plasma membranes from the homogenate of cerebellar granule cells differentiated in culture. It consisted in a centrifugation of the postnuclear fraction P2, on a Percoll gradient with formation of an upper and lower band. The upper band, upon centrifugation on 1 M sucrose, produced a light band lying on the top, that constituted the plasma membrane preparation. The upper band constituted the lysosome preparation. The plasma membrane preparation exhibited a 6-fold relative specific activity increase of Na+, K(+)-ATPase and 5'-nucleotidase, with negligible contamination by other subcellular markers; the lysosomal preparation exhibited a 30-fold relative specific activity increase of beta-galactosidase and beta-hexosaminidase, with virtually no contamination by other subcellular markers. Both the lysosome and plasma membrane preparations carried sialidase activity on MUB-NeuNAc and ganglioside GD1a. The sialidase activity on GD1a required the presence of Triton X-100 in both subcellular preparations; the sialidase activity on MUB-NeuNAc was markedly activated by albumin only in the lysosomes. The lysosomal sialidase had a unique optimal pH value, 3.9. The plasma membrane sialidase featured two values of optimal pH, one at 3.9, for both substrates and second at 5.4 and 6.0 for MUB-NeuNAc and GD1a, respectively. It is concluded that cerebellar granule cells differentiated in vitro possess one lysosomal sialidase and two plasma membrane sialidases, all of them active on ganglioside.     

Glycerylphosphorylcholine phosphodiesterase in rat liver. Subcellular distribution and localization in plasma membranes

1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5'-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5'-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a K(m) for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe(3+)).     

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ- 28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.     

A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation

We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.     

Isolation and partial characterization of chick brain synaptic plasma membranes.

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Separation of the microvillous (maternal) from the basal (fetal) plasma membrane of human term placenta: methods and physiological significance of marker enzyme distribution.

Plasma membranes from normal, full-term human placental trophoblast have been isolated by a new procedure. The method depends upon isopycnic zonal centrifugation using linear sucrose/Ficoll density gradients. Enrichment of plasma membrane marker enzymes with respect to trophoblast homogenate is found in two distinct peaks (designated B and D) of the fractionated effluent recovered from the rotor. Fraction B is enriched with membrane-bound alkaline phosphatase and 5'-nucleotidase, but not with (Na+, K+)-ATPase of F(-)-stimulated adenylate cyclase. It is suggested that this material is derived from the maternal-facing microvillous plasma membrane. Fraction D, enriched with (Na+, K+)-ATPase, F(-)-stimulated adenylate cyclase and, to a smaller extent, with 5'-nucleotidase and alkaline phosphatase is, by exclusion, proposed to be derived from the fetal-facing basal plasma membrane. Both plasma membrane fractions are shown to be free of appreciable contamination, using specific markers for endoplasmic reticulum, mitochondria, nuclei and lysosomes. The separation of the two membrane fractions is shown to depend both upon these membranes forming closed vesicles during homogenization and upon the buoyant densities of such vesicles differing in such a way that microvillous plasma membranes band at a lower density than basal plasma membranes. No separation of the membranes is achieved in gradients in which the vesicles are collapsed.     

The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On electron microscopy, the final plasma membrane fractions are judged to be freethe basis of of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5'-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

Wheat germ agglutinin is selectively transported to multivesicular bodies.

Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Enzyme profiles of mammalian bile.

The activities of subcellular marker enzymes in bile and liver homogenate from several mammalian species have provided information on the specificity of protein release during bile formation. The presence of significant amounts of the plasma membrane enzymes alkaline phosphodiesterase I and leucyl-beta-naphthylamidase in bile, in addition to alkaline phosphatase and 5'-nucleotidase, and the relative absence of intracellular enzymes lends support to the view that bile salt liberation from the hepatocyte is accompanied by a partial solubilization of the plasma (canalicular) membrane without extensive damage to the whole hepatocyte.     

Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.     

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.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Preparation and characterization of the lateral and basal plasma membranes of the rat intestinal epithelial cell

A technique is described for the isolation of a plasma-membrane fraction from the rat intestinal epithelial cell which is distinct from the microvillus membrane of that cell. The isolated fraction contains only about 0.2% of the sucrase activity in the original homogenate and negligible quantities of nuclear and mitochondrial membrane markers. It contains 12% of the total Na(+),K(+)-dependent adenosine triphosphatase and 7% of the alkaline phosphatase, with significant increments in specific activity of these enzymes. Multiple membrane preparations were highly reproducible with respect to the specific activities of the markers studied. The small intestine of one rat yields material containing about 1.3mg of protein. In addition an assay is described suitable for determining 5'-nucleotidase in the small intestine.     

Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.