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.     

Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity

Summary Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density pertubation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978.Membr. Biochem. 1: 177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979.Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochromec reductase, alkaline phosphatase, acid phosphatase, and galactosylstransferase.Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochromec reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochromec reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillovers by the adjacent, galactosyltransferase-rich population. This work emphasize the importance of multiple, physical criteria for purity in the isolation of subcellular components.     

Stimulation-associated redistribution of Na,K-ATPase in rat lacrimal gland

Summary To test the possibility that stimulation of secretion leads Na,K-ATPase to be recruited from cytoplasmic pools and inserted into basal-lateral plasma membranes, we surveyed the subcellular distributions of Na, K-ATPase in resting and stimulated fragments of rat exorbital lacrimal gland. After a two-dimensional separation procedure based on differential sedimentation and density gradient centrifugation, we defined sixdensity windows, which differ from one another in their contents of biochemical markers. The membranes equilibrating inwindow I could be identified as a sample of basal-lateral membranes; in resting preparations these membranes contained Na,K-ATPase enriched 16.6-fold with respect to the initial homogenates.Windows II throughVI contained various cytoplasmic membrane populations; these accounted for roughly 80% of the total recovered Na,K-ATPase activity. Thirty-minute stimulation with 10 m carbachol caused a 1.4-fold increase (P<0.05) in the total Na,K-ATPase content ofwindow I; this increase could be largely accounted for by a 1.7-fold decrease in the total Na,K-ATPase content ofdensity window V. Acid phosphatase activity also redistributed following stimulation, but it was recruited from a different source, and it was inserted into membranes equilibrating inwindows II andIII as well as into the membranes ofwindow I.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Prostaglandins E₁ and E₂ are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E₂ binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine β-hydroxylase). The only other fractions enriched in prostaglandin E₂ binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E₂ binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

Plasma membranes from rat Sertoli cells: purification and properties

A method is reported for preparing surface (plasma) membranes from rat Sertoli cells. The procedure is based upon homogenization in hypotonic buffer, extraction in a two-phase system, and sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by electron microscopy, enzyme markers, and functional activities associated with the membranes (binding of follicle-stimulating hormone [FSH] and production of cyclic adenosine 5'-monophosphate [cAMP]. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside of the membrane sheets. Marker enzymes for plasma membrane (5'-nucleotidase and alkaline phosphatase) showed more than 16- and 6-fold enrichment, respectively, and other enzymes showed that contamination by nuclei, mitochondria, endoplasmic reticulum, or cytosol was negligible. Binding of FSH was found to be specific, with KD 1.2 nM and the equivalent of 7500 sites per cell. This binding was enriched 20-fold compared to whole homogenate. Production of cAMP by membranes was increased by addition of FSH and by forskolin to the purified membranes in vitro.     

Isolation of the basal and lateral plasma membranes of rat kidney tubule cells.

A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.     

Renal brush-border-membrane vesicles prepared from newborn rats by free-flow electrophoresis and their proline uptake.

A method for the isolation of brush-border membranes from newborn-rat kidney, employing centrifugation and free-flow electrophoresis, is described. The composition and purity of the preparation was assessed by determination of enzyme activities specific for various cellular membranes. Free-flow electrophoresis resolves the newborn-rat renal membrane suspension into two populations of alkaline phosphatase-enriched brush-border membranes, designated 'A' and 'B', with the A peak also showing activity of (Na+ + K+)-stimulated ATPase, the basolateral membrane marker enzyme, whereas those of the B peak were enriched 11-fold in alkaline phosphatase and substantially decreased in (Na+ + K+)-stimulated ATPase activity. Membranes in the A peak showed a 7-fold enrichment of alkaline phosphatase, and (Na+ + K+)-stimulated ATPase activity similar to that of the original homogenate. Proline uptake employed to assess osmotic dependency revealed 7% binding of proline to the B vesicles and 31% to the A vesicles. This contrasts with 60% proline binding to vesicles prepared by centrifugation alone. Unlike vesicles from adult animals, proline uptake by B vesicles did not show an Na+-stimulated overshoot, but did exhibit an Na+-gradient enhanced rate of early proline entry. proline entry.     

Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants

Summary A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8mm CaCl₂ concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.     

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.     

Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : II. Enzymatic Characterization and Comparison with Other Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.     

Isolation of luminal and antiluminal membranes from dog kidney cortex.

Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na+ + K+)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Expression of an active Na,K-ATPase with an alpha-subunit lacking all twenty-three native cysteine residues

We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min(-)(1) versus 6655 min(-)(1)). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na(+), a 2-fold increase in K(m) for K(+), and no changes in K(m) for ATP. The distribution of alpha-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution.     

Enzymatic characterization of subcellular samples from gypsy moth larval midgut following differential centrifugation and fractionation on Percoll-sucrose gradient

A method is described for isolation of an enriched fraction of plasma membranes from gypsy moth (Lymantria dispar) larval midgut tissue. Following differential centrifugation of tissue homogenate, a microsomal sample is obtained and fractionated on a Percoll®-sucrose gradient that yields 2 distinct regions of high protein concentration: one enriched in plasma membranes, the other in mitochondrial membranes. The procedure is relatively rapid, being completed within approximately 5 h. Protein yields and accompanying specific activities are reported for marker enzymes used to indicate the presence of plasma membranes (leucine aminopeptidase and alkaline phosphatase), endoplasmic reticulum (NADPH-cytochrome c reductase), and mitochondria (succinate dehydrogenase). The apparent differences between the plasma membrane enriched fraction vs. brush border membrane vesicles prepared from insect midguts are discussed, as is the suitability of the plasma membrane enriched fraction for ATP-dependent calcium ion transport studies. © 1992 Wiley-Liss, Inc.     

Basal-lateral membranes from rabbit renal cortex prepared on a large scale in a zonal rotor

Basal-lateral membranes from the renal cortex of the rabbit were isolated by sucrose gradient centrifugation in a zonal rotor which allows for a large-scale preparation of these membranes. A heterogeneous population of membranes (P4) which contained 29% of the (Na+ + K+)-ATPase found in the homogenate of renal cortex was prepared by differential centrifugation. When pellet P4 was subjected to centrifugation in a sucrose gradient the activity of (Na+ + K+)-ATPase, a marker for basal-lateral membranes, could be separated from enzymatic markers of other organelles. The specific activity of (Na+ + K+)-ATPase was enriched 12-fold at a density of 1.141 g/cm3. Membranes (P alpha) contained in the (Na+ + K+)-ATPase-rich fractions consisted primarily of closed vesicles which exhibited probenecid inhibitable transport of rho-aminohippurate. These membranes did not exhibit Na+-dependent, phlorizin-inhibitable D-glucose transport. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from P alpha revealed at least six major protein bands with molecular weights of 91000, 81000, 73000, 65000, 47000 and 38000. A small fraction of total alkaline phosphatase found in the homogenate was found in pellet P4. Membranes containing this alkaline phosphatase activity were distributed widely over the gradient, with peak activity found at a density of 1.141 g/cm3. In contrast, when brush borders were subjected to gradient centrifugation under the same conditions as P4, alkaline phosphatase was found in a narrow distribution, with peak activity at a density of 1.158 g/cm3. The principle subcellular localization of the alkaline phosphatase found in P4 could not be determined unambiguously from the data, but the activity did not seem to be primarily associated with classical brush borders.     

Mutational analysis of alpha-beta subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane

The beta-subunit of the Na,K-ATPase is required to deliver functional alpha beta-heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the beta-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the alpha-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of beta-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by beta-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated beta-subunits and some disulfide bridge substitution mutants assemble with the alpha-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.     

Isolation of plasma membranes from neurons grown in primary culture

Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB3H4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the "wash" fractions were pooled and centrifuged at 37,000g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20- and 12-fold enriched in (Na+,K+)-adenosinetriphosphatase and 5'-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose:N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.     

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.