The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays. Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface.

Polydiacetylenic lipid membranes offer a general 'litmus test' for molecular recognition at the surface of a membrane. A concentration of 20 ppm of protein could be detected using polymerized thin films. The speed, sensitivity and simplicity of the design offers a new and general approach towards the direct colorimetric detection of a variety of different molecules.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.

Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as a stable 900-kDa complex. The serotype A 900-kDa complex is one of the forms of the toxin being used as a therapeutic agent for the treatment of various neuromuscular disorders. Previous experiments have demonstrated that the 900-kDa complex form of the toxin protects the toxin from the harsh conditions of the gastrointestinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the toxic (botulinum neurotoxin) component, the three species have been investigated with a series of biophysical techniques at the molecular level (dynamic light scattering, proteolysis, circular dichroism, pH incubations, and agglutination assays). These experiments were conducted under harsh conditions which mimic those found along the gastrointestinal tract. Separately, exposure to denaturing and proteolytic conditions degrades both the botulinum neurotoxin and the nontoxic component. In the 900-kDa complex, the botulinum neurotoxin is protected during exposure to the gastrointestinal environment and the nontoxic component is slightly modified. Surprisingly, the toxin protects the ability of the nontoxic component to agglutinate erythrocytes. Contrary to previous reports, the purified 900-kDa complex did not have agglutination ability until after exposure to the proteolytic conditions. These experiments provide new evidence and detail for the theory that the nontoxic component and the toxic component protect one another during exposure to harsh conditions, and a molecular model is presented for the passage of the toxin through the gastrointestinal tract.