THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.     

Evidence for a transition in the cortical membranes of Paramecium

Ever since the pioneering studies in the 1960s and 70s, the importance of order transitions for cell membrane functions has remained a matter of debate. Recently, it has been proposed that the nonlinear stimulus-response curve of excitable cells, which manifests in all-or-none pulses (action potentials (AP)), is due to a transition in the cell membrane. Indeed, evidence for transitions has accumulated in plant cells and neurons, but studies with other excitable cells are expedient in order to show if this finding is of a general nature. Herein, we investigated intact, motile specimens of the “swimming neuron” Paramecium. The cellular membranes were labelled with the solvatochromic fluorophores LAURDAN or Di-4-ANEPPDHQ. Subsequently, a cell was trapped in a microfluidic channel and investigated by fluorescence spectroscopy. The generalized polarization (GP) of the fluorescence emission from cell cortical membranes (probably plasma and alveolar membranes) was extracted by an edge-finding algorithm. The thermo-optical state diagram, i.e. the dependence of GP on temperature, exhibited clear indications for a reversible transition. This transition had a width of ~10–15 °C and a midpoint that was located ~4 °C below the growth temperature. The state diagrams with LAURDAN and Di-4-ANEPPDHQ had widely identical characteristics. These results suggested that the cortical membranes of Paramecium reside in an order transition regime under physiological growth conditions. Based on these findings, membrane potential fluctuations, spontaneous depolarizing spikes, and thermal excitation of Paramecium was interpreted.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

Transbilayer mapping of membrane proteins using membranes isolated on polylysine-coated polyacrylamide beads

Erythrocyte and HeLa cell plasma membranes were isolated on polylysinecoated polyacrylamide beads and the transbilayer disposition of their proteins was investigated.When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution.When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[³H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.     

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.     

Heterogeneity in the physical state of the exterior and interior regions of mycoplasma membrane lipids

The electron paramagnetic resonance spectra of spin-labeled fatty acid in intact mycoplasma cells and isolated membrane preparations have been compared. With Mycoplasma hominis and Acholeplasma laidlawii preparations, the freedom of motion of the spin-label was higher in labeled intact cells than in labeled isolated membranes but no differences could be detected between the labeled intact cells and membranes isolated from the labeled intact cells. It is proposed that the higher freedom of motion of the spin-label in the intact cells is due to a higher fluidity of the outer half of the lipid bilayer of mycoplasma membranes rather than to alterations in the structure of the membrane upon isolation.     

Light,Temperature, and Desiccation Effects on Photosynthetic Activity,and Drought-Induced Ultrastructural Changes in the Green Alga <Emphasis Type="Italic">Klebsormidium dissectum</Emphasis> (Streptophyta) from a High Alpine Soil Crust

Members of the cosmopolitan green algal genus Klebsormidium (Klebsormidiales, Streptophyta) are typical components of terrestrial microbiotic communities such as biological soil crusts, which have many important ecological functions. In the present study, Klebsormidium dissectum (Gay) Ettl &; Gärtner was isolated from a high alpine soil crust in the Tyrolean Alps, Austria. Physiological performance in terms of growth and photosynthesis was investigated under different controlled abiotic conditions and compared with ultrastructural changes under the treatments applied. K. dissectum showed very low light requirements as reflected in growth patterns and photosynthetic efficiency. Increasing temperatures from 5°C to 40°C led to different effects on respiratory oxygen consumption and photosynthetic oxygen evolution. While at low temperatures (5–10°C), respiration was not detectable or on a very low level, photosynthesis was relatively high, Reversely, at the highest temperature, respiration was unaffected, and photosynthesis strongly inhibited pointing to strong differences in temperature sensitivity between both physiological processes. Although photosynthetic performance of K. dissectum was strongly affected under short-term desiccation and recovered only partly after rehydration, this species was capable to survive even 3 weeks at 5% relative air humidity. K. dissectum cells have a cell width of 5.6?±?0.3 μm and a cell length of 8.4?±?2.0 μm. Desiccated cells showed a strongly reduced cell width (46% of control) and cell length (65% of control). In addition, in desiccated cells, fewer mitochondria were stained by DIOC₆, and damaged plasma membranes were detected by FM 1–43 staining. High-pressure freeze fixation as well as chemical fixation allowed visualizing ultrastructural changes caused by desiccation. In such cells, the nucleus and chloroplast were still visibly intact, but the extremely thin cell walls (75–180 nm) were substantially deformed. The cytoplasm appeared electron dense and mitochondria were altered. Although K. dissectum can be characterized as euryoecious species, all ecophysiological and ultrastructural data indicate susceptibility to desiccation. However, the steadily occurring fragmentation of filaments into smaller units leads to improved self protection and thus may represent a life strategy to better survive longer periods of drought in exposed alpine soil crusts.     

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{≈ 130 000}$) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight.To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer membrane surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.     

On the use of cells or membranes for receptor binding: Growth hormone secretagogues

Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K_d value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K_i values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays.     

Characterization of phosphoinositide kinases in normal and sickle anaemia red cells

PtdIns and PtdInsP kinases from normal erythrocyte (AA) membranes and sickle cell anaemia erythrocyte (SS) membranes have been characterized. PtdIns kinase was studied in native membranes under conditions in which PtdInsP kinase and PtdInsP phosphatase do not express any activity. Kinetic analysis of the AA and SS PtdIns kinases indicate similar K_m values for PtdIns and ATP but higher V_max values for SS PtdIns kinase. PtdInsP kinase was partially purified from erythrocyte ghosts by NaCl extraction. The kinetic parameters of PtdInsP kinase determined under these conditions were similar in AA and SS NaCl extracts. These data suggest the presence of some effector of PtdIns kinase in SS cell membranes, resulting in a greater activity of the enzyme. This leads consequently, to increase the PtdInsP pool and to activate PtdInsP kinase, in agreement with our previous observations of a greater [³²P]Pi incorporation in both polyphosphoinositides in SS cells relatively to AA cells.     

Distribution of cholesterol between the outer and inner halves of the lipid bilayer of mycoplasma cell membranes

Rapid kinetic studies of filipin binding to intact cells and isolated membranes were performed with a stopped-flow apparatus to determine the distribution of cholesterol in the outer and inner surfaces of mycoplasma membranes. The initial rates of association of filipin with cholesterol in Mycoplasma gallisepticum and Mycoplasma capricolum intact cells were slower than those obtained with isolated membrane preparations. Ratios of the second-order rate constants for filipin binding to cells relative to membranes indicate that cholesterol is distributed symmetrically in membranes of M. gallisepticum cells whereas in M. capricolum ～66% of the free cholesterol is localized in the outer half of the lipid bilayer.     

Metabolism of separated leaf cells: I. Preparation of photosynthetically active cells from tobacco

Suspensions of mesophyll cells, prepared from tobacco leaves by treatment with pectinase, fixed CO₂ by photosynthesis. The products of carbon assimilation were similar for both cells and intact tissue. The cells sustained a constant fixation rate for 20 to 25 hours. For optimal CO₂ fixation, enzymatic maceration of the tissue was accomplished in 0.8 m sorbitol, but photosynthesis was optimal in 0.6 m sorbitol at pH 7 to 7.5. A hypertonic environment during maceration, which results in cell plasmolysis, is essential to maintain intact plasmalemmas and hence photosynthetically active cells. For sustained CO₂ fixation, light intensities below 500 foot-candles were required. Higher light intensities (to 1000 foot-candles) gave high initial rates of CO₂ fixation, but the cells bleached and were inactive on prolonged incubation. At pH 7.0 the bicarbonate concentration at maximal velocity of CO₂ fixation was about 1.5 mm and the apparent Km for bicarbonate was 0.2 mm.     

Changes in secondary structure of poliovirus ribonucleic acid.

The incorporation of the fluorescent amine, dansyl cadaverine [N(5-aminopentyl)-5-dimethylamino-1-naphthalene sulfonamide], into the plasma membranes of intact cells was investigated. Using a fluorescent microscope, incorporation was observed when cultured mouse lymphoma (L1210) cells, cultured human fibroblasts and white cells from several sources were incubated in the presence of 0.1 mM dansyl cadaverine. While intact erythrocytes from several species did not incorporate the fluorescent amine, erythrocyte ghosts did. The uptake of dansyl cadaverine by L1210 cells was dependent upon the cell concentration, incubation time and temperature. Experiments designed to elucidate the structural requirements for fluorophor uptake demonstrated that, in addition to a hydrophobic dansyl group an extended straight hydrocarbon side chain with either an amino or hydroxyl group was necessary. The incorporated fluorophor was noncovalently associated with the cell membrane as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes and extraction of dansyl cadaverine labelled cells with choroform/methanol (2:1). These results indicate that dansyl cadaverine is incorporated into plasma membranes and suggest its potential usefulness as a new fluorescent probe in cell membrane studies.