Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by 125I/lactoperoxidase or 3H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.     

Human lymphocyte membrane proteins treated with neuraminidase

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.     

Isolation of the Ochromanas danica plasma membrane and identification of several membrane enzymes

Ochromonas danica cell homogenate can be fractionated by differential centrifugation into chloroplast, mitochondrial, ribosome, lysosomal, plasma membrane and soluble fractions. The plasma membrane fraction was further purified by discontinuous sucrose density gradient centrifugation and was found to be enriched 4–16-fold in the following enzymes: β-galactosidase, acid phosphatase, alkaline phosphatase, 5′-nucleotidase, and (Na⁺, K⁺)-ATPase. The role of plasma membrane phosphatase in the phosphate metabolism of plants is discussed.     

Changes in nuclear non-histone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells

Nuclei from colonic epithelial cells were isolated and fractionated by centrifugation in discontinuous sucrose density gradients. Nuclei differing in buoyant density differ in size, non-histone protein to DNA ratio, and capacity for DNA synthesis in vivo. They do not differ in histone content or in proportions of the major histone classes. The distribution of cell nuclei after density gradient centrifugation corresponds functionally to their histological localization in the colonic mucosa, as judged by the nuclear capacity for DNA synthesis in both normal and tumor tissues. The nuclei of colonic epithelial cells contain a heterogeneous set of non-histone proteins which change in total amount and in relative proportions during normal differentiation. The complement of nuclear proteins differs in normal intestinal epithelial cells and in colon tumors induced by the carcinogen, 1,2-dimethylhydrazine. There is a striking increase in the nuclear content of two major protein classes (of mol. wt ca 44 000 and 62 000) during carcinogenesis. The accumulation of these proteins in the nuclei of carcinogen-treated cells follows early, selective increases in their rates of synthesis.     

Cellular fractionation and isolation of the plasma membrane of Burkitt's lymphoma cells

A procedure for cellular fractionation and preparation of plasma membrane from a Burkitt's lymphoma cell line is described. This procedure involves homogenization with a Polytron in buffered isotonic sucrose, and separation of cellular fractions by differential and isopycnic centrifugation in sucrose. The isolated plasma membrane fraction contains 44% of the cellular cholesterol, 50% of the ouabain-sensitive (Na⁺ + K⁺)-ATPase activity, 43% of the γ-glutamyltranspeptidase activities and 16% of the phospholipid. This fraction contains only 3% of cellular protein and is contaminated with less than 4% of the total cellular activities of microsomal, lysosomal, mitochondrial, Golgi and soluble marker enzymes. The cholesterol : phospholipid molar ratio of the crude plasma membrane is 0.56. The membranes in this fraction are in the form of vesicles. Further purification of plasma membrane is achieved by sucrose density gradient centrifugation and results in a 25- to 30-fold enrichment of plasma membrane markers. Plasma membrane markers band in these gradients between 1.10 and 1.15 g/cm³.The distribution patterns in the cell fractions of 18 cellular constituents are quantitatively determined. Most constituents are found to distribute in a fashion consistent with the results obtained in other systems. Thymidine-5′-phosphodiesterase (phosphodiesterase I), esterase, nucleoside diphosphatase and glucose-6-phosphatase, however, are shown to be poor markers of membrane fractions in this system.Lactoperoxidase-catalyzed iodination was used to identify several plasma membrane proteins which are exposed at the surface. After separation of labeled polypeptides by sodium dodecyl sulfate gel electrophoresis, the predominant labeled protein was identified as the heavy chain of IgM. Several lesser labeled proteins were observed.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

The plasma membrane ATPase of Dictyostelium discoideum

During centrifugation of Dictyostelium membranes on sucrose and metrizamide gradients, an ATPase activity resistant to azide and molybdate but sensitive to diethylstilbestrol was found to copurify with the plasma membrane markers alkaline phosphatase and ¹²⁵I in cells surface-labelled by lactoperoxidase catalyzed iodination. This ATPase was enriched 50-fold in purified plasma membranes and could be separated from the mitochondrial ATPase on metrizamide gradients. The plasma membrane ATPase is very specific for ATP as substrate and Mg²⁺ as essential cofactor. Its pH optimum is 6.5 and it is inhibited by dicyclohexylcarbodiimide, diethylstilbestrol, vanadate, mercurials and Cu²⁺, but not by ouabain, molybdate, azide or oligomycin. It was not specifically affected by either monovalent cations or anions. These results suggest that the plasma membranes of Dictyostelium contain an ATPase similar to the proton-pumping ATPases recently identified in fungal and plant plasma membranes (Serrano, R. (1984) Curr. Top. Cell. Regul. 23, 87–126).     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Localization of proteins in the inner and outer membranes of Caulobacter crescentus

Cytoplasmic and outer membranes of Caulobacter crescentus were separated by isopycnic sucrose gradient centrifugation into two peaks with buoyant densities 1.22 and 1.14 g/cm³. These peaks were identified as outer and cytoplasmic membranes by the enrichment of malate dehydrogenase and NADH oxidase in the lower density peak and the presence of flagellin, a cell surface protein, in the heavier peak. The identity of the heavier peak as outer membrane was confirmed by labeling of cells with diazotized [³⁵S]sulfanilic acid, a reagent that does not penetrate intact cells. Under these conditions only outer membrane proteins were substituted by the sulfanilic acid. The distribution of proteins between the cytoplasmic and outer membranes were examined by the analysis of [³⁵S]methionine-labeled membranes by SDS-polyacrylamide and two-dimensional gel electrophoresis. These results showed that the inner and outer membranes contain approximately equal numbers of proteins, and that the distribution of these proteins between the two layers is highly asymmetric. Although many of the proteins could be assigned to one or the other membrane fraction, a number of the outer membrane proteins in the 32 000–100 000 molecular weight range frequently contaminate the inner membrane fractions. The implications of these results for membrane isolation and separation in C. crescentus are discussed.     

Peeling as a novel, simple, and effective method for isolation of apical membrane from intact polarized epithelial cells

Apical membrane of polarized epithelial cells is generally isolated by physicochemical methods, that is, precipitation with polyethylene glycol (PEG) or MgCl₂ followed by differential centrifugation or sucrose density gradient centrifugation. However, these protocols are considerably sophisticated and frequently accompanied by impurities (e.g., contaminations of basolateral membrane and intracellular organelles), particularly by inexperienced investigators. We have developed a simple and effective method for isolation of apical membrane from intact polarized renal tubular epithelial cells. On the basis of hydrous affinity and/or ionic interaction, the apical membrane could be efficiently peeled from the cells by four different materials—Whatman filter paper, nitrocellulose membrane, cellophane, and glass coverslip—all of which are available in most research laboratories. Phase-contrast and laser-scanning confocal microscopic examinations using anti-ZO-1 antibody showed that other parts of the cells, particularly tight junction complex, remained intact after peeling by all four of these surfaces. Western blot analyses of gp135 (apical membrane marker) and of Na⁺/K⁺-ATPase, LAMP-2, COX-4, and calpain-1 (markers of basolateral membrane, lysosome, mitochondria, and cytosolic compartment, respectively) revealed that peeling with Whatman filter paper and glass coverslip was most and second-most effective, respectively, without any contaminations from basolateral membrane and other intracellular organelles that could be detected in the samples isolated by peeling with nitrocellulose membrane and cellophane and by conventional methods (i.e., precipitation with PEG or MgCl₂ followed by differential centrifugation or sucrose density gradient centrifugation). Our physical method is very simple, easy to follow (even by inexperienced investigators), time-saving, and cost-effective with a higher efficiency (as compared with conventional methods) for isolation of apical membrane from polarized epithelial cells.     

Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition

The subcellular localization of the membrane-associated CTP:phosphocholine cytidylyltransferase was determined in Chinese hamster ovary cells in which the phospholipid composition had been altered by growth in the presence of N-methylethanolamine or treatment with phospholipase C. Cell homogenates were fractionated on Percoll density gradients, and marker enzyme activities were used to determine the location of the cellular membrane fractions. The peak of cytidylyltransferase activity occurred in the gradient at a density intermediate to that of the peaks of endoplasmic reticulum and plasma membrane markers. The profile of cytidylyltransferase activity most closely resembled that of the Golgi membrane marker; however, upon sucrose gradient centrifugation, the profile of the Golgi apparatus was very different from that of cytidylyltransferase. Differential centrifugation suggested a nuclear membrane association of the enzyme. Cytidylyltransferase was associated with a membrane fraction that sedimented when subjected to very low speed centrifugation (65 x g, 5 min). From Percoll gradient fractions, nuclei were identified by microscopy, and they migrated with cytidylyltransferase activity. The data are consistent with a localization of cytidylyltransferase in the nuclear membrane.     

Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Isolation and identification of Methylomonas methanica membranes

The crude membrane preparation of Methylomonas methanica was fractionated by sucrose density gradient centrifugation and in an aqueous dextran -- polyethylene glycol two-phase system. Fractions of a higher purity were prepared by sucrose density gradient centrifugation. Two subcellular fractions were isolated and characterized. One of them enriched in lipopolysaccharides was represented by the cell wall debris; the other possessing greater specific activities of the enzymes contained mainly intracytoplasmic membranes. The effect of various factors on the separation of membranes and on the specific enzyme activities was investigated.     

Ultraviolet light-induced crosslinking of HnRNA to informofer proteins in vitro

RNA-protein interaction in the 30S subunits of rat liver hnRNP has been studied by crosslinking of informofer proteins to hnRNA induced by UV irradiation.Irradiation of 30S particles with 254 nm UV light in doses of 1×10⁵ erg/mm² leads to the extensive crosslinking hnRNA to informofer proteins. The crosslinked material was analyzed either by resedimentation in a 15–30% sucrose gradient in the presence of 3 M guanidine-HCl and 1 M NaCl or by centrifugation in a Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl. The crosslinked complexes sedimented at about 25S in the sucrose gradient and proved to be heterogeneous in isopycnic centrifugation experiments. The proteins of the crosslinked complexes were analyzed by polyacrylamide gel electrophoresis. Proteins with M_r values of 70 000, 58 000, 43 000 and 40 000 appeared to be crosslinked with hnRNAs of the 30S particles.In the unirradiated 30S particles after centrifugation in the Cs₂SO₄ density gradient containing guanidine-HCl and sarkosyl two minor proteins were observed with M_r values of 70 000 and 58 000, banded in density zones characteristic for free RNA.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation

Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation.     

Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains.