Characterization of the plasma membrane of Mycoplasma laidlawii. III. The formation and aggregation of small lipoprotein structures derived from sodium dodecyl sulfate-solubilized membrane components (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1971
Reaggregation of solubilized membranes as a means for the fractionation of membrane proteins was tested with Acholeplasma laidlawii membranes. The dialysis of membranes solubilized by IO mM sodium sulfate against 20 mM Mg 2+ for I h, or against 5 mM Mg 2+ for 72 h, yielded reaggregated membranes exhibiting a different protein profile in polyacrylamide gels from that of the native membranes. The reaggregated membranes obtained under similar conditions from membranes solubilized by 20 mM sodium dodecyl sulfate did not significantly differ from the native membranes in protein composition. The membrane material solubilized by 20 mM sodium dodecyl sulfate met several criteria for complete membrane solubilization as distinct from the material solubilized by IO mM sodium dodecyl sulfate, which contained minute membrane fragments sedimentable at IOOOOO × g for I h with a higher content of certain membrane proteins. It was stipulated that the incorporation of these fragments into the reaggregated membranes was responsible for the difference in protein composition between them and the native membranes. No significant differences in lipid types could be detected between membranes reaggregated under different dialysis conditions. It is concluded that the incorporation of the various membrane proteins and lipids into the reaggregated membranes is not selective. The ultrastructure of the reaggregated membranes is discussed in light of these and other findings and the hypothesis is put forward that the reaggregated membranes essentially consist of a bimolecular leaflet of lipid covered with protein on both sides.
Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis
1988
Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (³âµS)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (³H)palmitate-labeled cells showed that approximately 20 proteins of this
Characterization of Membrane Components of the Flask-shaped Mycoplasma Mycoplasma mobile
Microbiology, 1988
The cell membrane of Mycopfasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only onethird of cellular proteins and had a density of 1.14 g ml-l. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45,25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol :phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho-and glycolipids were detected. The phospholipid fraction consisted of a major de novosynthesized phosphatidylglycerol (-63 % of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (-90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated :unsaturated fatty acids ratio.
Membrane lipids of Mycoplasma fermentans
FEMS Microbiology Letters, 1994
Membranes of Mycoplasma fermentans, incognitus strain, were isolated by a combination of osmotic lysis and sonication. Analysis of membrane lipids revealed, in addition to free and esterified cholesterol, six major polar lipids dominated by a de novo synthesized compound (compound X), which accounts for 64% of the total lipid phosphorus. Compound X was labeled by palmitate, but not by oleate. Mass spectrometry and gas liquid chromatography analyses of compound X revealed two molecular species with molecular masses of 1048 and 1076 representing, a dipalmitoyl-and a stearoyl-palmitoyl-glycerodiphosphatidylcholine. Compound X has the ability to stimulate human monocytes to secret TNFa and to enhance the fusion of small unilamellar vesicles with MOLT-3 lymphocytes.
Are mycoplasma membrane proteins affected by variations in membrane fatty acid composition?
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1972
and on dissolution in 1% sodium dodecyl sulphate, spectra analysis indicated the presence of the membrane cytochromes. The organic solvent and aqueous phases (i.e. I and 3) were, of course, invariably present, but Phases 2 and 4 were variable. The solvent phase up to, but not including, the interfacial material was carefully removed with a chilled Pasteur pipette. The aqueous phase was adjusted to 3 ml with distilled water and two further extractions performed. After a third extraction, a Pasteur pipette was introduced beneath the interface and the aqueous phase removed, taking care not to disturb the floating, interfacial layer or the pellet. The aqueous phase was dialyzed at o °C against several changes of buffer, its final volume measured and samples taken for protein, asp radioactivity and enzymatic determinations. It was imperative to keep the temperature well controlled during extraction, as marked variability in results was observed if temperature fluctuations were allowed to occur. Radioactivity measurements Organic solvent phase extracts were each dried under a stream of N 2 gas and taken up into 3 ml of chloroform. Samples of o.o5-o.1 ml were applied to ~rhatman glass fiber paper discs wb.ich were dried and placed on planchets and the 3~p determined in a Nuclear Chicago gas-flow detector. Radioactivity in the aqueous phases was similarly determined, Total phospholipid radioactivity was determined by extracting 3 ml of an aqueous suspension of membranes with chloroform-methanol (I :2, v/v) by the method of Bligh and Dyer 11. Two extractions were performed, the two organic phases combined and dried under a stream of N 2 gas. The lipid was dissolved in 3 ml of chloroform and kept at-20 °C until required. Great difficulty was encountered in attempting to disperse the pellets and interfacial layers. The residual 3zp remaining in these combined phases was therefore calculated by substracting the 32p in the aqueous and solvent phases from the total as determined by the chloroformmethanol extractions. Chromatography Paper chromatography of the lipid extracts was performed on silica-gel loaded paper (Whatman no. SG-8I), using the solvent system of Wuthier lz (chloroformmethanol-diisobutyl ketone-acetic acid-water (45:15:20:30:4, by vol.)). Lipids were located by staining with Rhodamine 6G (o.oo12 % w/v) in distilled water, the spots cut out, placed on planchets and the radioactivity counted. Samples from the origin, solvent front and intermediate zones were also counted. Identification of the phospholipids was made by simultaneous chromatography of purified M. lysodeikticus phospholipids kindly supplied by Dr. August De Siervo. Polyacrylamide gel dectrophoresis Polyacrylamide gel electrophoresis was carried out using standard procedures employed in this laboratory13, la. Samples were adjusted to 1-2 mg pro¢ein/ml and o.15-o.18 ml applied to each column. Chemical and enzymatic assays Protein was determined by the method of Lowry et al. 15. ATPase (EC 3.6.1.3) was assayed according to Mufioz et al. 16 and NADH dehydrogenase (EC 1.6.99.3) was assayed according to Nachbar and Salton 17.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1971
I. The purified glycolipid haptens of Mycoplasma pneumoniae were reaggregated with Acholeplasma laidlawii membrane proteins. The process consisted of the solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glyeolipids in 2o mM sodium dodecyl sulfate, and dialysis of the solution separately or in mixtures against 2o mM Mg 2+. 2. The reaggregated material collected by centrifugation of the dialyzed solution of lipid-depleted A. laidlawii membrane proteins consisted of amorphous clumps, while the reaggreated M. pneumoniae glycolipids consisted of "myelin-like" globules and sheets composed of lamellae with a mean center-to-center distance of 37 A. The reaggregated material of a mixture of lipid-depleted A. laidlawii membrane proteins and M. pneumoniae glycolipids contained, in addition to the amorphous clumps representing reaggregated proteins and the "myelin-like" structures representing reaggregated glycolipids, also long membranous sheets having a triplelayered structure with a mean center-to-center distance of the dense lines of 54 A. The appearance and dimensions are closely similar to those of the original or reagregated A. laidlawii membranes. 3. It is suggested that these membrane-like structures are formed by the association of A. laidlawii membrane protein and M. pneumoniae glycolipids, and that these "hybrid" structures are responsible for the increased antigenicity of the reaggregated glycolipids.
Is the vertical disposition of mycoplasma membrane proteins affected by membrane fluidity?
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979
The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-toprotein ratio through treatment with chloramphenicol. The lactoperoxidasemediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.
Binding of proteins to mycoplasma membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1973
1. Isolated Acholeplasma laidwalii membranes were capable of binding large quantities of the basic proteins cytochrome c and lysozyme (up to 0.5 mg per mg membrane protein), usually smaller amounts of n-butanol-solubilized A. laidlawii membrane proteins and still less of bovine serum albumin. 2. Removal of about 70% ofA. laidlawiimembrane proteins by pronase digestion increased and the removal of lipids decreased the binding capacity of the membrane, so that the lipids appear to provide most of the binding sites for the soluble proteins. 3. Isolated membranes bound about twice as much cytochrome c and lysozyme as membranes of intact cells, implying that about half the binding sites for these proteins are exposed on the outer membrane surface. 4. The binding of the proteins to the membranes depended on temperature, pH and ionic strength of the medium. The binding of albumin and butanol-solubilized membrane proteins increased considerably at acid pH values while that of cytochrome c and lysozyme was only slightly affected. The membrane-bound cytochrome c and lysozyme, but neither the albumin nor the butanol-solubilized proteins, could be almost quantitatively released by 1 M NaCI, showing electrostatic bonds to be responsible for the binding of the basic proteins. 5. Butanol-solubilized A. laidlawii membrane proteins could bind to Mycoplasma mycoides var. mycoides membranes so as to produce "hybrid" membranes having antigenic properties of both membrane types. The possibility of binding proteins from the growth medium to the mycoplasma membrane and its implications regarding immunological characterization are discussed.