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Biomedical subjects

A Helenius

Publications and source records attributed to A Helenius.

At least 163 records · Page 9Linked to original sources
PubMed1978

Formation of protein micelles from amphiphilic membrane proteins.

The membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis, the Semliki Forest virus spike proteins, and the Sendai virus glycoproteins have each been isolated as soluble protein aggregates that are virtually free of lipid and detergent. The sedimentation coefficients of the complexes were 18 S, 29 S, and 43 S, respectively. Mixed aggregates containing both the virus glycoproteins and the penicillinase could also be formed. Such protein micelles may serve a number of useful purposes in membrane research.

Antibodies↗
PubMed1978

Reconstitution of purified detergent-soluble HLA-A and HLA-B antigens into phospholipid vesicles.

Purified detergent-soluble human histocmpatibility antigens (HLA-A and HLA-B) were reconstituted into phospholipid vesicles by mixing the protein and lipid together in the presence of either octylglucoside (octyl-beta-D-glucopyranoside) or deoxycholate and removing the detergent by dialysis. The resulting preparation consisted of lipid vesicles containing all or most of the added protein. The protein in the vesicles was antigenically active, as demonstrated by specific binding to anti-beta2-microglobulin IgG-Sepharose beads and by specific inhibition of alloantibody and complement-mediated cytotoxicity. Protein incorporated into vesicles at a protein/phospholipid ratio of 1:10 showed an asymmetric distribution of the HLA-A and HLA-B molecules, with virtually all of the antigens oriented facing the external medium. Cleavage experiments with proteases showed that the molecule was attached to the vesicle membrane via the COOH terminus, consistent with its proposed structure in intact cellular plasma membranes. Electron micrographs of the vesicles showed 50-60 A knobs on the outer surface similar to structures observed for other membrane proteins. HLA-A and HLA-B could also be incoporated into vesicles together with Semliki Forest virus membrane proteins. The resulting preparations should be useful in defining the molecular interactions involving HLA-A and HLA-B antigens in the immune response.

Cholesterol↗
PubMed1978

Human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest virus.

The proteins coded for by the HLA-A and HLA-B loci in man and the H-2K and H-2D loci in mice were identified as cell surface receptors for Semliki Forest virus. This conclusion is based on the following observations: (i) Water-soluble octamers of viral coat proteins inhibit the complement-dependent cytotoxicity of antibodies directed against H-2K and H-2D antigens in mouse cells. (ii) Isolated detergent-soluble HLA-A and HLA-B antigens reconstituted in lipid vesicles inhibit the binding of viral proteins to human cells (as do the water-soluble antigens to a lesser extent). (iii) Reconstituted HLA-A and HLA-B vesicles interact in solution with Semliki Forest virus (or with vesicles containing viral spike proteins), as demonstrated by coprecipitation with antisera. (iv) Complexes between viral spoke proteins and HLA-A and HLA-B antigens or H-2K and H-2D antigens can be isolated from the cell surface by utilizing affinity chromatography or immunoprecipitation.

Animals↗
PubMed1977

Charge shift electrophoresis: simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution.

Seventeen hydrophilic proteins and five amphiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton X-100), a mixture of anonionic and an anionic detergent (Triton X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylammonium bromide). The electrophoretic mobility of the hydrophilic proteins was unaffected in the three detergent mixtures. However, the mobility of the amphiphilic proteins shifted anodally in the Triton X-100-deoxycholate system and cathodally in the Triton X-100-cetyltrimethylammonium bromide system when compared to the mobility in Triton X-100 alone. The detergent-induced shift in mobility provides a simple, rapid, and sensitive method for distinguishing between hydrophilic and amphiphilic proteins.

Electrophoresis, Agar Gel↗
PubMed1977

Reconstitution of Semliki forest virus membrane.

The spike glycoproteins of the Semliki forest virus membrane have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis method. The detergent used was beta-D-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by hydrophobic peptide segments, as in the viral membrane. Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation.

Centrifugation, Density Gradient↗
PubMed1976

Semlike Forest virus membrane proteins. Preparation and characterization of spike complexes soluble in detergent-free medium.

After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95-10(4) and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as "rosette"-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.

Crystallization↗
PubMed1976

Solubilization of the Semliki Forest virus membrane with sodium deoxycholate.

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.

Binding Sites↗
PubMed1975

Solubilization of the Semliki Forest virus membrane with sodium dodecyl sulfate.

The dissociation of Semliki Forest virus induced by increasing concentrations of the anionic detergent sodium dodecyl sulfate was studied using density gradient centrifugation. Detectable binding to the virus started well below the critical micellar concentration of the detergent and increased thereafter with increased detergent concentration. At 4 degrees there were about 11,000 binding sites per virus particle with an average association constant of about 10-5 M-1. The extent of virus dissociation could be controlled both by the detergent concentration and by the temperature. At 4 degrees only disruption ("lysis") of the virus membrane could be observed. At 20 degrees most of the membrane was solubilized into lipoprotein complexes, and the nucleocapsid dissociated into RNA and protein. Complete delipidation of the viral membrane proteins was achieved at 30 degrees at a detergent concentration still below the critical micellar concentration.

Binding Sites↗