Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes.
The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides GM1, GM3, GD1a, GD1b, GT1b and asialo-GM1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for GM1 (Ka = 1.8.10(8)M-1), a lower one for asialo-GM1 (Ka = 1.0.10(7)M-1) and no affinity for GM3. The C-fragment of tetanus toxin binds to ganglioside GD1a, GD1b and GT1b containing membranes with similar affinity (Ka approximately 10(6)M-1), while no binding was observed with GM3. Pertussis toxin binds to membranes containing the ganglioside GD1a with a binding constant of Ka = 1.6.10(6)M-1, but only if large amounts (40 mol%) of GD1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to GD1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to GD1a.
Janshoff
A
A
Institut für Biochemie, Westfälische Wilhelms-Universität, Münster, Germany.
Steinem
C
C
Sieber
M
M
el Bayâ
A
A
Schmidt
M A
MA
Galla
H J
HJ
D002772:Cholera Toxin / Q000378:metabolism*; D017097:Electric Impedance; D005732:Gangliosides / Q000378:metabolism*; D006046:Gold; D008051:Lipid Bilayers / Q000378:metabolism*; D008567:Membranes, Artificial; D037342:Pertussis Toxin; D010713:Phosphatidylcholines; D011791:Quartz; D013057:Spectrum Analysis / Q000379:methods; D013744:Tetanus Toxin / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D005732:Gangliosides; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D013744:Tetanus Toxin; D010566:Virulence Factors, Bordetella; D011791:Quartz; D006046:Gold; D002772:Cholera Toxin; D037342:Pertussis Toxin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine