Reconstitution of the Functional Membranes from Chloroplasts with the Deficient Crista Membranes from Mitochondria

The basic mechanisms of phosphorylation of chloroplasts and mitochondria are identical. The identity may be proved by membrane combination. There are two ways to get the combination as shown in figure 1. One way is, as previously reported, to combine deficient membranes from chloroplasts with ctista membranes from mitochondria and the reconstituted membranes thus obtained greatly enhance photophosphorylated activities. The other way, i.e., to combine deficient crista membranes with thylakoid membranes, has also been successful, as shown in this paper. The reconstituted membranes obtained in this way can carry out oxidative phosphorylation in the dark as well as shown in Table 2. There are some relationship between the ATP formation from oxidative phosphorylation of reconstituted membranes and the protein of deficient crista membranes added, as shown in Table 3. When the quantity of combined chloroplast membranes is kept constant, the amount of ATP formation varies, within certain limits, with the amount of deficient crista membranes as shown in Table 3. But the reconstituted oxidative phosphorylation activity of membranes formed by combinating thylakoid with deficient crista membranes is lower than reconstituted photophosphorylation activity of combination in the opposite direction, i.e. by combinating deficient thylakoid membranes and crista membranes of mitochondria (compare Table 4 and 3).     

Heritable variation in erythrocyte membranes of mice with muscular dystrophy

Several physical and chemical characteristics of erythrocyte membranes from dystrophic mice differ from those of controls. In this study it is postulated that there is a heritable antigenic characteristic in erythrocyte membranes of dystrophic mice that is associated with muscular dystrophy. Accordingly, antisera were prepared in goats against erythrocyte membranes from dystrophic mice. These antisera discriminated between membranes from control mice and membranes from three different strains of dystrophic mice. Preliminary data on carrier detection are consistent with the hypothesis that the antigen is manifest in membranes of carriers.     

Characteristics of the fine structure of embryonic membranes in the spiny-headed worms of the class Eoacanthocephala, by an example of the spiny-headed worm species Neoechinorhynchus crassus

Five embryonic membranes are found in the spiny-headed worm species Neoechinorhynchus crassus. Four embryonic membranes are analogous to the membranes in all other spiny-headed worms studied, and one membrane is additional. The last is situated between the external and second membranes and is characteristic only for some species of the class Eoacanthocephala. Terminology of the embryonic membranes in connection with their origin and possible functional significance is discussed.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Transbilayer organization of membrane cholesterol at low concentrations: Implications in health and disease

Cholesterol is an essential and representative lipid in higher eukaryotic cellular membranes and is often found distributed nonrandomly in domains in biological membranes. A large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content. However, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol such as the endoplasmic reticulum or inner mitochondrial membranes. In this review, we have traced the discovery and subsequent development of the concept of transbilayer cholesterol dimers (domains) in membranes at low concentrations. We have further discussed the role of membrane curvature and thickness on the transbilayer organization of cholesterol. Interestingly, this type of cholesterol organization could be relevant in cellular sorting and trafficking, and in pathological conditions.     

Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.     

A novel method of preparation of small intestinal brush border membrane vesicles by polyethylene glycol precipitation

A novel method of brush border membrane vesicle (BBMV) preparation from the small intestinal mucosa using polyethylene glycol (PEG) precipitation has been presented. This preparation is compared with calcium-precipitated BBMVs in marker enzyme enrichment, contamination by other subcellular membranes, transport of glucose, and lipid composition. PEG-precipitated BBMVs are comparable with calcium-precipitated membranes in all parameters except lipid composition and thiol content. PEG-precipitated membranes have more phosphatidylcholine and phosphatidylethanolamine and less lysophosphatidylcholine and lysophosphatidylethanolamine as compared to calcium-precipitated membranes. Diacylglycerol and triacylglycerol content are also high in PEG-precipitated membranes. Alteration in lipid composition indicate the possible activation of lipase and phospholipase by calcium during BBMV preparation, which is not seen in PEG precipitation. Thiol content is almost double in PEG-precipitated membranes as compared to calcium-precipitated membranes. These results indicate that PEG can be used for the preparation of BBMVs in native form from the intestine without any alteration in their structural components, and these membranes show comparable transport activity.     

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.     

Cholesterol Function in Plasma Membranes from Ectotherms: Membrane-Specific Roles in Adaptation to Temperature

SYNOPSIS. Cholesterol is an essential component in the plasmamembranes of animals with multiple effects on the physical propertiesof membranes including membrane order (fluidity), phase behavior,thickness, and permeability. Cholesterol also affects functionalattributes of cell membranes such as the activities of variousintegral proteins. Because cholesterol provides rigidity tofluid phase membranes, it is a likely candidate to counter someof the temperature-induced perturbations in membrane order thatwould otherwise be experienced by animals that live at variedbody temperatures. If cholesterol contributes to homeoviscousadaptation (HVA), more cholesterol is likely to be present inplasma membranes from warm-bodied animals than from cold-bodiedanimals. This prediction is generally supported by studies examiningcholesterol contents in membranes from endothermic and ectothermicanimals. Comparisons of cholesterol levels in temperature acclimated(oracclimatized) ectotherms reveal an increase in cholesterolwith temperature, no change in cholesterol content, or an increasein cholesterol with a decrease in temperature. These differentpatterns largely represent tissue and regional differences inthe membranes (membrane domains). The membranespecific natureof the cholesterol response to temperature is likely to arisefrom the multiplicity of the effects that cholesterol exertson membranes, as well as the heterogenous nature of plasma membranes.These factors also allow cholesterol to perform more than asingle role in temperature adaptation of plasma membranes inanimals.     

Fatty-acid composition of lipids and physicochemical characteristics of membrane fractions and the membrane of endoplasmic reticulum of thymus and Pliss' lymphosarcoma

The paper is concerned with composition of neutral lipids and phospholipids, the regularity of lipid bilayer and structural reorganization of plasma membranes, and membranes of smooth and rough cell reticulum of thymus and Pliss lymphosarcoma are studied at linear and stationary growth phase. No qualitative differences are found in the fatty-acid composition of lipid membranes in normal and tumour cells. In plasma membranes of phospholipids and in membranes of smooth reticulum of tumour cells the unsaturated lipid component increases in the process of growth, the cholesterin/phospholipids ratio decreases, fluidity of the lipid bilayer diminishes and structural heterogeneity of these membranes rises while in membranes of rough reticulum the saturation of lipids increases, but the cholesterin/phospholipids ratio does not change. The temperatures of structural reorganization also does not change, which evidences for a less structural lability of membranes of rough reticulum as compared with other membranes.     

Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasizing lymphoma (Eb) and its metastasizing variant (ESb)

Plasma membrane fragments from two variants of a murine lymphoma, Eb and ESb, with different metastatic capacity were investigated. Plasma membranes were isolated from tumor cells recovered from the peritoneal cavity. They differed in their lipid composition, indicating a more fluid state of the plasma membranes derived from the highly metastatic tumor line ESb. Extracellular membrane vesicles could be isolated from the ascites of the tumor-bearing mice. The shedding capacity of ESb cells was much higher than that of Eb cells. The extracellular membranes by chemical analysis and the measurement of marker enzymes proved to be derived from the plasma membranes. However, they differed from the plasma membranes from which they were derived in several aspects: (i) the lipid to protein ratio was diminished; (ii) the activities of some plasma membrane-associated enzymes were lower while other were identical in plasma membranes and extracellular membranes; (iii) the content of saturated fatty acids in phopholipids was enhanced in extracellular membranes. These effects were more pronounced in the highly metastasizing tumor line ESb. It is thus concluded that shedding of extracellular membranes is not a random process. The biochemical differences found in the plasma membranes and the extracellular membranes of the two tumor lines are discussed with respect to the different metastatic capacity of the tumors.     

Jejunal and ileal d-glucose transport in isolated brush border membranes

d-Glucose transport was investigated in isolated brush border membranes from small intestine. The transport properties of membranes from upper jejunum were compared with those from terminal ileum. The jejunal membranes accumulated d-glucose to a greater extent than the ileal membranes when supplied with energy in the form of a NASCN gradient. This difference is behavior is similar to that of the more intact epithelial preparations and suggests that the isolated membranes actually reflect the state present in intact cells. Ileal membranes transported d-glucose about two to three times slower than the jejunal ones, which can partially explain the lower sugar accumulation.     

Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1- pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre- activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.     

Bull sperm plasma and acrosomal membranes: Fluorescence studies of lipid phase fluidity

Bull sperm plasma and outer acrosomal membranes have been isolated by discontinuous sucrose density gradient centrifugation and Ca²⁺-ATPase activity has been determined for both the membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization studies show that the lipid phase of the sperm plasma membranes is more fluid than the lipids of the outer acrosomal membranes. Approximately, a three fold increase in the cholesterol content has been found in the outer acrosomal membranes as compared to that in the plasma membranes.     

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.     

Isolation and characterization of subcellular membranes of Entamoeba invadens

A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (beta-glycerophosphate) was associated mainly with phagolysosmal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not beta-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 mumol/mumol, respectively). Microsomes were relatively poor in cholesterol (0.39 mumol/mumol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of spingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process.     

Single-cell unroofing: probing topology and nanomechanics of native membranes

Cell membranes separate the cell interior from the external environment. They are constituted by a variety of lipids; their composition determines the dynamics of membrane proteins and affects the ability of the cells to adapt. Even though the study of model membranes allows to understand the interactions among lipids and the overall mechanics, little is known about these properties in native membranes. To combine topology and nanomechanics analysis of native membranes, I designed a method to investigate the plasma membranes isolated from a variety of single cells. Five cell types were chosen and tested, revealing 20% variation in membrane thickness. I probed the resistance of the isolated membranes to indent, finding their line tension and spreading pressure. These results show that membranes isolated from neurons are stiffer and less diffusive than brain cancer cell membranes. This method gives direct quantitative insights on the mechanics of native cell membranes.     

Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.     

The regulation of adenylate cyclase activity in turkey erythrocyte membranes by forskolin

The mechanisms by which forskolin stimulates adenylate cyclase activity in turkey erythrocyte membranes and is influenced by manganese and Gpp(NH)p were studied. Forskolin-dependent adenylate cyclase activity in particulate turkey erythrocyte membranes is enhanced following preincubation of membranes with isoproterenol and GMP (cleared membranes). In contrast, solubilization of turkey erythrocyte membranes, previously cleared, renders them relatively refractory to forskolin but not to Gpp(NH)p. Whereas adenylate cyclase activity due to the simultaneous presence of forskolin and Mn2+ in particulate turkey erythrocyte membranes is additive, their copresence becomes synergistic after solubilization. The apparent Kact for forskolin activation of adenylate cyclase is not influenced by clearance or by the presence of Mn2+ in particulate turkey erythrocyte membranes. Following solubilization, the Vmax for forskolin-dependent adenylate cyclase activation determined in the presence of Mn2+ is also independent of clearance. Forskolin activation of turkey erythrocyte adenylate cyclase appears to be influenced at sites in addition to the catalytic unit.     

Phospholipid composition of nuclear membranes and cell nuclei of rat liver and hepatoma 27]

Phospholipid composition of nuclei and nuclear membranes from rat liver and hepatoma-27 were investigated. Hepatoma nuclei and nuclear membranes were found to contain cardiolipin which was absent in the same fractions of rat liver. In the nuclei and nuclear membranes of hepatoma the content of sphingomyelin was higher and that of lecithin is lower than in the corresponding subcellular fractions of rat liver. The content of acid phospholipids was much higher in hepatoma nuclear membranes than those of the normal liver. The data obtained show that the general trend of lipid dedifferentiation which earlier was demonstrated for various membranes of different tumor cells is observed also in the case of nuclear membranes.