The distribution of surface antigens during fractionation of mouse liver plasma membranes.
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ABSTRACT:
SUBMITTER: Gurd JW
PROVIDER: S-EPMC1174324 | biostudies-other | 1972 Nov
REPOSITORIES: biostudies-other
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Project description:1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.
| S-EPMC1185506 | biostudies-other
Project description:1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an (125)I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate-1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5'-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate-Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.
| S-EPMC1165901 | biostudies-other
Project description:The lateral heterogeneity of rat liver plasma membranes was examined by fragmentation and fractionation by counter-current distribution in an aqueous two-phase polymer system. The distribution pattern was analysed by plotting the relative specific activities of marker components against each other. By this analysis asialo-orosomucoid receptors were found in a domain separated from domains containing 5'-nucleotidase and leucine aminopeptidase by another domain devoid of these markers. 5'Nucleotidase and leucine aminopeptidase resided in adjacent but separate domains. The experimental data were compared with corresponding plots of markers in model membranes. The model membranes yielded plots of different shapes depending on marker distribution and fragment size. This method of analysis should be useful for examining the lateral heterogeneity also of other membranes.
| S-EPMC1148713 | biostudies-other
Project description:1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5'-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5'-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a K(m) for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe(3+)).
| S-EPMC1178596 | biostudies-other
Project description:1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.
| S-EPMC1177683 | biostudies-other
Project description:Borrelia burgdorferi is the causative agent of Lyme borreliosis, which is the most common tick-borne human disease in Europe and North America. Currently, the diagnosis of Lyme borreliosis is based on serological tests allowing indirect detection of anti-Borrelia antibodies produced by patients. Their main drawback is a lack of sensitivity in the early phase of disease and an incapacity to prove an active infection. Direct diagnostic tests are clearly needed. The objectives of this study were to produce tools allowing sensitive detection of potential circulating Borrelia antigens and to evaluate them in a mouse model. We focused on two potential early bacterial makers, the highly variable OspC protein and the conserved protein FlaB. High-affinity monoclonal antibodies were produced and used to establish various immunoassays and western blot detection. A very good limit of detection for OspC as low as 17 pg/mL of sample was achieved with SPIE-IA. In infected mice, we were able to measure OspC in plasma with a mean value of 10 ng/mL at 7 days post-inoculation. This result suggests that OspC could be a good blood marker for diagnosis of Lyme borreliosis and that the tools developed during this study could be very useful.
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Project description:Surface modification on microporous polyethylene (PE) membranes was facilitated by plasma polymerizing with two hydrophilic precursors: ethylene oxide vinyl ether (EO1V) and diethylene oxide vinyl ether (EO2V) to effectively improve the fouling against mammalian cells (Chinese hamster ovary, CHO cells) and proteins (bovine serum albumin, BSA). The plasma polymerization procedure incorporated uniform and pin-hole free ethylene oxide-containing moieties on the filtration membrane in a dry single-step process. The successful deposition of the plasma polymers was verified by Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS) analyses. Water contact angle measurements and permeation experiments using cell and protein solutions were conducted to evaluate the change in hydrophilicity and fouling resistance for filtrating biomolecules. The EO1V and EO2V plasma deposited PE membranes showed about 1.45 fold higher filtration performance than the pristine membrane. Moreover, the flux recovery reached 80% and 90% by using deionized (DI) water and sodium hydroxide (NaOH) solution, indicating the efficacy of the modification and the good reusability of the modified PE membranes.
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Project description:Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.
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Project description:Copper isotopes may prove to be a useful tool for investigating bacteria-metal interactions recorded in natural waters, soils, and rocks. However, experimental data which attempt to constrain Cu isotope fractionation in biologic systems are limited and unclear. In this study, we utilized Cu isotopes (?(65)Cu) to investigate Cu-bacteria interactions, including surface adsorption and intracellular incorporation. Experiments were conducted with individual representative species of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, as well as with wild-type consortia of microorganisms from several natural environments. Ph-dependent adsorption experiments were conducted with live and dead cells over the pH range 2.5-6. Surface adsorption experiments of Cu onto live bacterial cells resulted in apparent separation factors (?(65)Cu(solution-solid) = ?(65)Cu(solution) - ?(65)Cu(solid)) ranging from +0.3‰ to +1.4‰ for B. subtilis and +0.2‰ to +2.6‰ for E. coli. However, because heat-killed bacterial cells did not exhibit this behavior, the preference of the lighter Cu isotope by the cells is probably not related to reversible surface adsorption, but instead is a metabolically-driven phenomenon. Adsorption experiments with heat-killed cells yielded apparent separation factors ranging from +0.3‰ to -0.69‰ which likely reflects fractionation from complexation with organic acid surface functional group sites. For intracellular incorporation experiments the lab strains and natural consortia preferentially incorporated the lighter Cu isotope with an apparent ?(65)Cu(solution-solid) ranging from ~+1.0‰ to +4.4‰. Our results indicate that live bacterial cells preferentially sequester the lighter Cu isotope regardless of the experimental conditions. The fractionation mechanisms involved are likely related to active cellular transport and regulation, including the reduction of Cu(II) to Cu(I). Because similar intracellular Cu machinery is shared by fungi, plants, and higher organisms, the influence of biological processes on the ?(65)Cu of natural waters and soils is probably considerable.
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Project description:Cell fractionations and other biological separations frequently require several steps. They could be much more effectively done by filtration, if isoporous membranes would be available with high pore density, and sharp pore size distribution in the micro- and nanoscale. We propose a combination of two scalable methods, photolithography and dry reactive ion etching, to fabricate a series of polyester membranes with isopores of size 0.7 to 50 μm and high pore density with a demonstrated total area of 38.5 cm2. The membranes have pore sizes in the micro- and submicro-range, and pore density 10-fold higher than track-etched analogues, which are the only commercially available isoporous polymeric films. Permeances of 220,000 L m-2 h-1bar-1 were measured with pore size 787 nm. The method does not require organic solvents and can be applied to many homopolymeric materials. The pore reduction from 2 to 0.7 μm was obtained by adding a step of chemical vapor deposition. The isoporous system was successfully demonstrated for the organelle fractionation of Arabidopsis homogenates and could be potentially extended to other biological fractionations.
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