New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.     

The Molecular Basis for Membrane – Cytoskeleton Association in Human Erythrocytes

Spectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane-spanning glycoproteins and with intramembrane particles. The presence of a specific, high-affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin-depleted inside-out vesicles. A 72,000 M_r proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 M_r polypeptide has been identified as the precursor of the spectrin-binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane-spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.     

Preparation of red-cell-membrane cytoskeletal constituents and characterisation of protein 4.1

A new and rapid method is described for the preparation of protein 4.1, the protein which modulates the interaction between spectrin and actin in the membrane cytoskeleton of the red cell. The method is based on the dissociation of purified membrane cytoskeletons in concentrated Tris at neutral pH, followed by gel filtration in the same medium. This procedure also yields spectrin and actin, as well as the fourth cytoskeletal constituent, protein 4.9, in relatively pure form, and ankyrin. Protein 4.1 is monomeric under our conditions of solvent and protein concentration, with a relative molecular mass, as determined from sedimentation equilibrium, of about 78 000; its sedimentation coefficient and Stokes' radius are those of a globular, though somewhat asymmetric or flexible molecule. It forms a strong complex with F-actin and spectrin. Protein 4.9 is also recovered in active form, and will bind strongly to F-actin.     

The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.     

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained.The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal.The yield of vesicles sealed to K⁺ was maximal at pH 4 and accounted for about one third of the total vesicle volume.The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K⁺.As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO₂²⁺.Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane.After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.     

Induction of serotonin release from mast cells by lymphocyte activators is dependent upon implantation of lymphocyte plasma membrane components.

Plasma membranes (PM) isolated from mouse splenic lymphocytes were successfully fused to rat basophilic leukemia (RBL) cells using intact Sendai virus virions (SV). A two-step procedure was used in which SV were first fused with the PM to create PM + SV vesicles; the vesicles formed were then incubated with the RBL cells. Insertion of lymphocyte PM into the RBL cell's membrane endows a high rate of serotonin secretion upon stimulation of the implanted RBL cells with antimouse Ig antibodies or with concanavalin-A. The results of the present work clearly suggest that activation signals can be delivered via implantation of foreign membrane preparation containing specific receptors, thus rendering the target cells susceptible to stimulation by specific reagents.     

Inducible synthesis of beta-galactosidase in disrupted spheroplast of Escherichia coli

A membrane preparation obtained from osmotic lysate of spheroplasts of Escherichia coli cells showed an activity of synthesizing beta-galactosidase which was dependent upon oxidative phosphorylation. The synthesis was inhibited by the addition of actinomycin D or of chloramphenicol. The beta-galactosidase synthesized in the membrane preparation was completely released into the medium, while that synthesized in the spheroplasts and intact cells remained within the cells. The minimum concentration of the inducer, methyl-beta-d-thiogalactoside, required for the induction of beta-galactosidase was 5 x 10(-5)m for intact cells, 3 x 10(-4)m for spheroplasts and 1 x 10(-3)m for membrane preparation. Incorporation of labeled glucose into insoluble components in membrane preparation was extremely low compared with that in intact cells or in spheroplasts. Based on these and other observations, the nature of this membrane preparation is discussed in relation to the structure of E. coli cells.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Interaction of Salmonella typhimurium with phospholipid vesicles. Incorporation of exogenous lipids into intact cells.

Incubation of intact cells of Salmonella typhimurium with bilayer phospholipid vesicles results in significant transfer of vesicle lipids to the cells. The transfer requires Ca2+ or spermine, and is dependent on time, temperature, the concentration and composition of the vesicles, and the nature of the cellular lipopolysaccharide. The process results in bulk transfer of vesicle lipids to the cells rather than reciprocal molecular exchange between vesicles and the outer membrane. All components of mixed lipid vesicles, including cholesteryl oleate and lipopolysaccharide, are transferred to the cells in a ratio similar to that of the donor vesicles. The properties of the transfer process are consistent with direct fusion of vesicles with the outer membrane of the cell.     

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles

The binding of human erythrocyte ankyrin (band 2.1) to the erythrocyte membrane has been characterized by reassociating purified ankyrin with ankyrin-depleted inside-out vesicles. Ankyrin reassociates at high affinity with a limited number of protease-sensitive sites located only on the cytoplasmic side of the erythrocyte membrane. Depleting the vesicles of band 4.2 does not affect their binding capacity. A 45,000-dalton polypeptide derived from the cytoplasmic portion of band 3 competitively inhibits the binding of ankyrin to inside-out vesicles. Although the bulk of band 3 molecules appear to have the potential for binding ankyrin, nly a fraction of the band 3 molecules in native membranes or in reconstituted liposomes actually provides accessible high affinity ankyrin binding sites.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

Kinetics and regulation of the ankyrin-band 3 interaction of the human red blood cell membrane

In an attempt to identify potential regulatory mechanisms for erythrocyte membrane-cytoskeletal interactions, the kinetics and pH dependence of the band 3-ankyrin interaction were investigated. Association of 125I-ankyrin with KI-stripped inside-out erythrocyte membrane vesicles was found to proceed in two kinetic phases. The initial, fast phase (t1/2 approximately 15-30 min) involved predominantly the binding of ankyrin to low affinity sites (KD approximately 130 nM) in a pH-dependent manner. The apparent pKa values describing this reversible pH dependence (7.2 +/- 0.1 and 9.2 +/- 0.1) defined states of band 3 with high, moderate, and no capacity to bind ankyrin (in order of increasing pH). Since the cytoplasmic domain of band 3 also exists in 3 distinct conformational states characterized by apparent pKa values of 7.2 and 9.2, it was hypothesized that the reversible structural equilibrium in band 3 could influence ankyrin binding. The second or slow phase of ankyrin binding to band 3 involved the conversion of low to high affinity sites (KD approximately 13 nM). This phase, which was largely temperature and pH independent, required roughly an order of magnitude longer to reach completion than the fast phase. Unfortunately, even though the slow phase could be cleanly separated from the fast phase at low pH, insufficient data were available to formulate a physical interpretation of its origin. Significantly, however, even after completion of the slow phase under the most quantitative binding conditions identified, a maximum of only 26% of the band 3 was found to bind ankyrin in situ. Although higher ankyrin-band 3 stoichiometries may be achievable with the isolated cytoplasmic fragment of band 3, we interpret the above 1:4 stoichiometry to suggest that the tetramer of band 3 constitutes the predominant ankyrin binding oligomer of band 3 on the membrane.     

ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with ¹²⁵I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.     

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.