Studies on plasma membranes

Summary Plasma membranes and lysosomes were isolated from rat liver and assayed for sialidase activity with gangliosides and sialyllactose as substrates. Plasma membrane and lysosomal activities differed in substrate preference, heat stability, inhibition by Cu²⁺,K _m values and pH dependence. It is concluded that plasma membranes and lysosomes contain different sialidases. Hepatoma plasma membranes also exhibited sialidase activity towards the two substrates.     

Studies on plasma membranes

Summary Plasma membranes were isolated from rat and mouse livers, one rat hepatoma (and its subline) and two mouse hepatomas, and their lipid class compositions were determined. Lipids accounted for 30 to 35% of the dry weight of the membranes of livers and mouse hepatomas, and for 45% in the case of rat hepatoma-subline. Of the total lipids of rat-liver plasma membranes, 60% consisted of phospholipids, the corresponding values for mouse-liver and rat-hepatoma plasma membranes amounting to 55% and for both mouse-hepatoma plasma membranes to about 50%. The free cholesterol and cholesteryl ester contents of all hepatoma plasma membranes were significantly increased as compared with normal. Evidence is presented that the increase of free cholesterol was not a preparative artefact. The major phospholipid classes in all plasma membranes were phosphatidyl choline, sphingomyelin, phosphatidyl ethanolamine and phosphatidyl serine. The relative proportions in each plasma membrane species could differ appreciably, the mouse- and rat-liver membranes showing the closest resemblance. Possible reasons for (a) the higher level of phosphatidyl serine as compared with published values, and (b) the wide divergencies which may be found among the phospholipid profiles of rat-liver plasma membranes reported by other authors, are presented. Cardiolipin was absent from liver plasma membranes, but some could be found in the hepatoma membranes due to mitochondrial contamination. No consistent phospholipid profile characterized hepatoma as distinct from liver plasma membranes, nor did the hepatoma data-including plasmalogens-resemble the few available data on other hepatomas.     

NADH oxidase of plasma membranes

NADH oxidase is a cyanide-resistant and hormone-responsive oxidase intrinsic to the plasma membrane of both plant and animal cells. The activity has many unique characteristics that distinguish it from other oxidases and oxidoreductases of both organelles and internal membranes and from other oxidoreductases of the plasma membrane. Among these are resistance to inhibition by cyanide, catalase, superoxide dismutase, and phenylchloromer-curibenzoate. Activity is stimulated by hormones and growth factors and inhibited by quinone analogs such as piericidin, the flavin antagonist atebrin, and growth inhibiting gangliosides such as G_M3. In marked contact to the NADH-ferricyanide oxidoreductase of the plasma membrane, the NADH oxidase is activated by lysophospholipids and fatty acids, products of phospholipase A₂ action, in a time-dependent manner suggestive of stabilization of an activated form of the enzyme. The hormone-responsive NADH oxidase of the plasma membrane is not a peroxidase and may function as a terminal oxidase to link transfer of electrons from NADH to oxygen at the plasma membrane. The functional significance of the NADH oxidase of the plasma membrane is unknown but some relationship to growth or growth control is indicated. In both animal and plant plasma membranes, the oxidase is activated by growth factors and hormones to which the cells or tissues of origin have functional hormone or growth factor receptors. In addition, substances that inhibit the oxidase, the associated transmembrane reductase or both, inhibit growth. In transformed cells and tissues, the hormone and growth factor responsiveness of the NADH oxidase is reduced or absent. With human keratinocytes which exhibit an increased sensitivity to the anti-proliferative action of both retinoic acid and calcitriol, the NADH oxidase of the plasma membrane is strongly inhibited by these agents and shows the same increased sensitivity. If transfer of electrons from NADH to oxygen across or within the eukaryotic plasma membrane is an important aspect of growth or growth control, then the hormone- and growth factor-responsive NADH oxidase associated with the plasma membrane could be of fundamental importance. Because of its low basal activity, stimulation by growth factors and hormones, and the inhibition of growth in direct proportion to inhibition of the oxidase, the activity is a candidate as a rate-limiting step in the growth process. Completely unknown is the mechanism whereby NADH oxidization and growth or growth control may be coupled. This, together with further characterization of the activity and the mechanism of loss of control with neoplastic transformation, represent important challenges for future investigations.     

Surface activity of plasma membranes

Plasma membranes of rabbit intestinal smooth muscle cells manifest low surface activity on the boundary of the electrolyte-air phases. This activity undergoes essential changes if the electrolyte surface is covered with the lecithin monolayer. According to the experimental data, the interaction of plasma membranes with the lecithin monolayers is hydrophobic in nature and essentially depends on the density of molecular packing of phospholipids in the monolayer and on the status of the membrane preparation. A possible mechanism of the formation of the monolayer patterns from cell membranes is discussed.     

Interaction of smooth muscle plasma membranes with flat lipid membranes

The method of implantation of smooth muscle cells from plasma membranes (PM) of the rabbit intestine into flat lipid membranes (FLM) is described. The method is based on the pretreatment of PM vesicles with asolectin liposomes in the ratio that provides the activation of membrane ATPases. Thus modified FLM possesses channel conductivity.     

Ca-ATPase of human myometrium plasma membranes

We determined and characterized the Mg2+-dependent, Ca2+-stimulated ATPase (Ca-ATPase) activity in cell plasma membranes from the myometrium of pregnant women, and compared these characteristics to those of the active Ca2+-transport already demonstrated in this tissue. Similarly to the Ca2+-transport system, the Ca2+-ATPase is Mg2+-dependent, stimulated by calmodulin, and inhibited by vanadate. The Km for Ca2+ activation is 0.40 microM, very similar to that found for active calcium transport, i.e. 0.25 microM. Consequently, this Ca2+-ATPase can be responsible for the active calcium transport across the plasma membranes of smooth muscle cells.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Aquaporins of plasma membranes of epithelial cells

The early 90s have brought us a discovery of a new class of membrane proteins--aquaporins with a function of transmembrane water channels. Being genetically closed proteins aquaporins are members of a large family of channel-forming proteins called MIPs (major intrinsic proteins). All aquaporins, except AQP4, are mercury-sensitive. Many aquaporins have been cloned and identified. Polyclonal antibodies grown against some of them promoted numerous studies of aquaporin localization and distribution in animal and plant tissues. Up to the present, 10 and 2 aquaporins have been described in mammalian and amphibian epithelial tissues, respectively. One of described aquaporins, AQP2, whose localization is confined to kidney collecting duct principal cells, has been found to be a hormone-depending water channel. The insertion of apical vesicles bearing AQP2 was shown to be regulated by vasopressin, meanwhile all other aquaporins are inserted into the plasma membrane constitutively. There is a vast evidence showing that the integrity of microtubules is necessary for both pathways of aquaporin insertion. AQP2 is important for normal kidney functioning and AQP2 mutations cause water-balance disorders. On the contrary, the AQP1 mutations are not accompanied by any evident clinical pathology. This review is focused on a discussion of the data so far available on aquaporin distribution in different animal tissues.     

Micrometer-scale domains in fibroblast plasma membranes

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.     

Glucose transport by uterine plasma membranes

Uterine plasma membrane preparations were obtained by centrifugation on discontinuous sucrose gradients. The specific activity of the plasma membrane marker 5'-nucleotidase was increased 10-fold while the specific activity of glucose-6-phosphatase was increased 3-fold. Electron microscopy showed mainly closed vesicles having diameters mainly in the range of 0.1 to 0.4 micron and an absence of other recognizable organelles such as mitochondria. D-Glucose transport was inhibited by sulfhydryl reagents, phloretin, and cytochalasin B. Uptake was prevented at high osmotic pressures. The Km of glucose transport was 12.2 +/- 1.1 mM. Studies of the inhibition of [3H]cytochalasin B binding by D-glucose indicated that the value of the Kd of the cytochalasin B-transporter complex was larger than 1 microM. These data demonstrate the potential usefulness of these preparations in the study of glucose transport in rat uterus and its control by steroid hormones.     

Cholesterol asymmetry in synaptic plasma membranes

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.     

Hyaluronate is synthesized at plasma membranes

The hybrid cell B6 line, which synthesizes large amounts of hyaluronate as the predominant glycosaminoglycan, was grown in the presence of [3H]glucosamine. The [3H]hyaluronate has a high molecular weight and was excluded by Sephacryl S-1000. After disruption of the cells the [3H]hyaluronate could further be elongated by incubation with UDP-GlcNAc and UDP-[14C]GlcA, yielding a hybrid molecule of hyaluronate labelled with [3H]GlcNAc and [14C]GlcA. Treatment of the cells with hyaluronidase before disruption eliminated the large [3H]hyaluronate and elongation of nascent chains in vitro commenced from low-molecular-weight chains. Thus nascent hyaluronate chains were degraded extracellularly by hyaluronidase and were therefore synthesized at the inner side of plasma membranes and extruded to the cell surface.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.