Separation of lipids from proteins and cell membranes by disc electrophoresis

Saltón, M. R. J.; Schmitt, Margreth D.

doi:10.1016/s0006-291x(67)80019-6

Cited by 37 publications

(4 citation statements)

References 3 publications

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“…At higher concentrations of detergent, similar to those used in the present study, most of the membrane material was found as micellar complexes containing protein, lipid and detergent (Helenius & Soderlund, 1973;Simons et al, 1973). At yet higher concentrations of detergent Engelman et al (1967), Salton & Schmitt (1967) and Helenius & Simons (1971) have shown that dissociation of membrane proteins from lipids takes place. Before this occurs, however, at intermediate concentrations of detergents, discrete complexes of proteins and lipids together with detergent molecules exist in solution.…”

Section: Discussionsupporting

confidence: 75%

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

Butters¹,

Hughes²

1974

Biochemical Journal

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1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.

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Section: Discussionsupporting

confidence: 75%

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

Butters¹,

Hughes²

1974

Biochemical Journal

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“…Disc electrophoresis of toxin-treated spherules. Salton and Schmitt (15) reported that mixtures of lipids and proteins could be effe-tively separated by disc-gel electrophoresis in the presence of deoxycholate (DOC). The lipid fraction moved as a complex, with the DOC ahead of the tracking dye, while the proteins were retained and separated in the separating gel.…”

Section: Resultsmentioning

confidence: 99%

“…A possible mechanism of hemolysis which can account for the results presented in this study, and for those indicating an enzymatic mechanism as FIG. 15. Fragments of osmotically prepared human erythrocyte ghiosts after reaction with HP a-toxinl.…”

Section: Discussionmentioning

confidence: 99%

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

1968

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Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal a-toxin indicated the presence of a lytic factor other than a-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified a-toxin (HP a-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure a 12S-toxin. The interaction of HP a-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The a 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An a 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the a 12S molecule. Lipid monolayer experiments showed that HP a-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP a-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to a-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to a 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.

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“…Some resolution of M. laidlawii membranes into protein and lipid was achieved when the membranes were dissociated with SDS and subjected to gradient centrifugation using a "sandwich technique" . Complete separation of membrane lipid from protein was also achieved by polyacrylamide gel electrophoresis of M. Iysodeikticus membrane preparations to which deoxycholate has been added just prior to electrophoresis (Salton and Schmitt, 1967b). Wallach (1969) has also reported that at high concentrations of SDS (2 %) membrane proteins go into solution as SDS-complexes which can be separated by polyacrylamide gel electrophoresis.…”

Section: A Molecular Interactions 43mentioning

confidence: 99%

The Bacterial Membrane

Saltón

1971

Biomembranes

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Separation of lipids from proteins and cell membranes by disc electrophoresis

Cited by 37 publications

References 3 publications

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

The Bacterial Membrane

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