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S Bhakdi

Publications and source records attributed to S Bhakdi.

At least 235 records · Page 13Linked to original sources
PubMed1983

Generation of leukotrienes and lipoxygenase factors from human polymorphonuclear granulocytes during bacterial phagocytosis and interaction with bacterial exotoxins.

The generation and release of lipoxygenase factors and leukotrienes from human polymorphonuclear granulocytes is demonstrated during bacterial phagocytosis and interaction with bacterial exotoxins (alpha-toxin, enterotoxin, lipase from Staph. aureus; Streptolysin O; cytotoxin from Pseudomonas aeruginosa). The leukotrienes released during stimulation exert chemotactic properties for human neutrophils and guinea pig eosinophils (leukotriene B4) and show the characteristic profile of slow reacting substance activity which is induced by leukotriene C4, D4 and E4. The toxin induced spasmogenic activity obtained from human PMNs was inhibited in the presence of the SRS-antagonist FPL 55712. The generation of lipoxygenase factors is also demonstrated by autoradiography using 14C arachidonic acid prelabelled granulocytes.

Animals↗
PubMed1983

[Structure and mode of action of bacterial toxins].

This paper provides an overview of the mechanisms by which bacterial exotoxins damage cells of the host organism. The principles of cellular attack are schematically described using typical representants of the various toxin groups as examples. Attention is drawn to structural and functional analogies existing among the toxins, and to related phenomena of cell damage by bacterioicins and the mammalian complement system. The development of immunotoxins illustrates the potential importance of basic research in toxinology and immunology to modern medicine.

Animals↗
PubMed1982

Hydrophilic-amphiphilic transition of the terminal SC5b-8 complement complex through tryptic modification: biochemical and ultrastructural studies.

the SC5b-8 complex of human complement is a hydrophilic molecule of mol. wt 800,000-850,000 that is seen in the electron microscope as an elongated, straight or curved structure of 50-55 nm total length and 8-9 nm width. Tryptic attack on the fluid-phase complex exposes lipid-binding surfaces on the molecule. The trypsinized complex can be incorporated into liposomal lipid bilayers, and the majority of protein is then viewed as ill-defined, larger tufts projecting exterior to the liposomal membrane. These tufts possibly represent clusters of a unit lesion, which consists of two diverging projections, each approximately 25 nm in length. The two projections are possibly joined to each other to give the membrane-bound complex a shape akin to that of an incomplete funnel. Analyses by SDS-polyacrylamide gel electrophoresis show that the polypeptide subunits C5b, C7 and C8 beta entirely resist tryptic degradation in both SC5b-8 and SC5b-9 complement complexes. Limited proteolysis of C6, C8 alpha gamma and C9, and extensive degradation of the S-protein are effected by trypsin. The results are compatible with the concept that proteolytic cleavage of the S-protein in SC5b-8 and SC5b-9 is the cause of the trypsin-dependent, hydrophilic-amphiphilic transition of the terminal, fluid-phase complement complexes.

Alpha-Globulins↗
PubMed1982

Terminal membrane C5b-9 complex of human complement: transition from an amphiphilic to a hydrophilic state through binding of the S protein from serum.

The membrane-damaging C5b-9(m) complex of complement is a cylindrically structured, amphiphilic molecule that is generated on a target membrane during complement attack. Isolated C5b-9(m) complexes are shown here to possess the capacity of binding a protein, termed "S"-protein, that is present in human plasma. Binding of this protein apparently shields the apolar surfaces of C5b-9(m), since the resulting "SC5b-9(m)" complex is hydrophilic and no longer aggregates in detergentfree solution. Dispersed SC5b-9(m) complexes exhibit an apparent sedimentation coefficient of 29S in sucrose density gradients, corresponding to a molecular weight of approximately 1.4 million. SDS PAGE analyses indicate binding of 3-4 molecules of S-protein per C5b-9(m) complex. These data are consistent with a monomer nature and molecular weight of 1-1.1 million of the C5b-9(m) complex. Ultrastructural analysis of SC5b-9(m) shows preservation of the hollow cylindrical C5b-9(m) structure. Additional material, probably representing the S-protein itself, can be visualized attached to the originally membrane-embedded portion of the macromolecule. The topography of apolar surfaces on a molecule thus appears directly probed and visualized through the binding of a serum protein.

Complement System Proteins↗
PubMed1981

A simple method for isolating specific antibodies to complement components.

A simple procedure for isolating specific antibodies to complement components C3, C4 and C5b-9 from whole rabbit antisera or immunoglobin preparations is described. Antisera are first reacted with complement-treated sheep erythrocyte membranes. Immunoglobulins are eluted from the washed membranes with acetic acid at pH 2.4, and further purified by one absorption/desorption step on Protein A Sepharose. The final protein preparations contain greater than 90% rabbit immunoglobins, as assessed by quantitative immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. Binding assays with radioiodinated protein preparations indicate a content of specific antibodies in the range 5-55% of total protein. Since there is virtually no non-specific binding of protein to control cells or membranes, the immunoglobulin preparations are well suited for use in sensitive immunoassays for complement components.

Animals↗
PubMed1981

Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer.

The hydrodynamic properties of the detergent-solubilized, terminal membrane complex of serum complement components C5-C9 [C5b-9(m)] were studied to obtain an estimate of its molecular weight. In a solution of Triton X-100/deoxycholate, the protein complex binds 17% Triton X-100 and 11% deoxycholate by weight. The sedimentation coefficient of the protein-detergent complex is 26 S as determined by sucrose density gradient ultracentrifugation, and gel filtration indicated a molecular radius of 11 nm. It was ascertained by electron microscopy that these hydrodynamic parameters apply to mono-dispersed C5b-9(m) complexes, which were observed as nonaggregated, hollow protein cylinders and were identical to the complement "lesions" formed on target membranes. The calculated molecular weight of the protein-detergent complex is approximately 1,286,300 to which the protein moiety contributes approximately 1,000,000. The results indicate that the C5b-9(m) complex formed on biological membranes is a monomer entity of the C5-C9 complement components.

Complement Membrane Attack Complex↗
PubMed1981

Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles.

Native staphylococcus aureus alpha-toxin is secreted as a hydrophilic polypeptide chain of Mr 34,000. The presence of deoxycholate above the critical micellar concentration induced the toxin monomers to self-associate, forming ring or cylindrical oligomers. The oligomers were amphiphilic and bound detergent. In deoxycholate solution, the protein-detergent complexes exhibited a sedimentation coefficient of 10.4 S. A Mr of 238,700 was determined by ultracentrifugation analyses at sedimentation equilibrium. Because quantitative detergent-binding studies indicated a protein/detergent ratio of approximately 5:1 (wt/wt), the protein moiety in each protein-detergent complex was determined to be approximately Mr 200000, corresponding to a hexamer of the native molecule. The amphiphilic toxin hexamers were ultrastructurally indistinguishable from the cytolytic, annular toxin complexes that form on and in biological target membranes. They bound lipid and could be incorporated into artificial lecithin lipid vesicles. The transition of toxin protein molecules from a hydrophilic monomer to an amphiphilic oligomer through self-association has thus been shown to be inducible solely through contact of the native protein molecules with an appropriate amphiphilic substrate.

Bacterial Toxins↗
PubMed1981

On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin.

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.

Animals↗
PubMed1981

Fluid-phase SC5b-8 complex of human complement: generation and isolation from serum.

A rapid, simple method for isolating SC5b-9 generated in human serum, and SC5b-8 generated in C9-depleted serum is described. The procedure leads to approximately 30% recovery of either complex and consists of polyethyleneglycol precipitation followed by 1 DEAE ion exchange chromatography and a sucrose density gradient ultracentrifugation. The SC5b-8 complex is a water-soluble macromolecular of alpha-electrophoretic mobility. Apart from the absence of C9, it exhibits an identical SDS-gel electrophoresis polypeptide pattern as SC5b-9. A sedimentation coefficient of 19 to 20S and an effective molecular radius of approximately 10 nm were determined by sucrose density gradient centrifugation and gel filtration, respectively. From these values, a m.w. of 800 to 850,000 is tentatively assigned to the SC5b-8 complex. The S-protein was not dissociable from either SC5b-8 or SC5b-9 through treatment with desoxycholate and no alterations in the hydrodynamic properties of either complex were discernible after action of this detergent.

Alpha-Globulins↗
PubMed1980

Monitoring of detergent binding to amphiphilic proteins by means of micelles containing the hydrophobic dye Sudan Black B.

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, or erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [3H]Triton X-100.

Animals↗
PubMed1980

The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel.

C5b-9(m) complexes were incorporated into lecithin liposomes and subjected to proteolysis in the presence of DTT to remove the externally oriented annulus. Liposomes were recovered that selectively carried the membrane-bound, thin-walled cylindrical portion of the C5b-9(m) complex. The presence of DTT during proteolysis enhanced peptide bond cleavage in the C5b-9(m) complex. All C5-C9 components were degraded to lower m.w. fragments. A protease-resistant, but hydrophilic 85 to 86,000-dalton polypeptide derivative of C5, possibly representing the C5 beta-chain, was recovered in the fluid phase. This component is not intimately associated with the target lipid bilayer. Immunochemical analyses yielded evidence for the existence of minor C5-C9 antigenic determinants on the membrane-bound C5b-9(m) residue. SDS polyacrylamide gel electrophoreses of liposomes carrying the C5b-9(m) residues revealed the persistence of at least six major polypeptides of approximately m.w. 50,000, 45,000, 40,000, 38,000, 20,000, and 16,000. The data are interpreted to indicate that multiple protease-resistant polypeptide chains derived from several terminal C components participate in formation of the trans-membrane C channel.

Antigens↗
PubMed1980

Re-incorporation of the terminal C5b-9 complement complex into lipid bilayers: formation and stability of reconstituted liposomes.

The formation and stability of lecithin liposomes carrying re-incorporated C5b-9(m) complexes prepared through a detergent-dialysis procedure was studied. Confluent aggregates of phospholipid and protein formed at low initial lipid-protein ratios (1:1, w/w), higher lipid-protein ratios (e.g. 5:1, w/w) were required for formation of lipid vesicles with recognizable bilayer structure. The unfractionated preparations comprised a heterogeneous population of vesicles that sedimented according to their lipid-protein content to varying positions upon centrifugation in CsCl density gradients. The vesicles were stable and C5b-9(m) complexes did not detach from the membranes during centrifugation through CsCl. They also resisted elution from the bilayer by treatment with salt solutions of low or high ionic strength, or of high pH. The results indicate anchorage of C5b-9(m) in the membrane through apolar interactions, akin to that of an integral membrane protein, and are compatible with the channel concept of complement lysis.

Centrifugation, Density Gradient↗
PubMed1979

Evidence for a two-domain structure of the terminal membrane C5b-9 complex of human complement.

Lipid vesicles carrying the purified membrane C5b-9 complex [C5b-9(m)] of complement were analyzed immunochemically and in the electron microscope after treatment with a combination of trypsin and alpha-chymotrypsin. Under reducing conditions, the externally oriented annulus was removed. The remaining part of the C5b-9(m), representing approximately half of the total mass of the macromolecular complex, was visualized in the electron microscope as a hollow cylindrical structure with walls of 1-nm thickness. This structure remained tenaciously attached to the lipid bilayer, projecting 8-9 nm from the external membrane surface into the aqueous environment. Cleavage of C5b-9(m) by proteolysis and reduction resulted in a sharp reduction of tis antigenic determinants. One hydrophilic protease-resistant C5 derivative was released from the membrane and recovered in the fluid phase. The membrane-bound residue almost totally lacked antigens precipitable with antisera to C5, C6, C9, and C5b-9(m).

Complement C5↗
PubMed1979

Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement.

Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion.

Antigens↗
PubMed1978

Molecular nature of the complement lesion.

The principle molecular event leading to membrane perturbation by complement is the assembly of the terminal five serum complement components (C5b-C9) into a macromolecular C5b-9 complex on the target membrane [Müller-Eberhard, H.-J. (1975) Ann. Rev. Biochem. 44, 697--723]. The present communication reports on the ability of purified C5b-9 complexes isolated from target membranes to become reincorporated into artificial lipid vesicles. The data indicate that the complex is a vertically oriented, hollow, cylindrical macromolecule possessing lipid-binding regions that enable one terminus to penetrate into the lipid bilayer. A transmembrane pore appears to be created at the attachment site of the C5b-9 complex.

Animals↗