(118) Assay Card

General Information
Summary Kalata B1 and B2 cause leakage of dye-containing POPC vesicles.
Condition Fluorescence emission spectra were measured between 460?600 nm on a Perkin-Elmer luminescence spectrometer with a scan speed of 140 nm/min. An excitation wavelength of 480 nm and a quartz cuvette with an optical pathwidth of 4 mm was used. The excitation and emission slit-widths were set to 4 nm. Experiments were performed at 25 C. The initial fluorescence signal, Fo (when vesicles are intact, i.e., no leakage), was measured and each run was started by the addition of 50 L of a peptide solution. Fluorescence spectra were measured every minute for 20 min before the addition of 10% Triton X-100 solution, whereby the vesicles are completely perturbed giving rise to the formation of mixed micelles and yielding a fluorescence signal of maximum intensity, Fx. Control experiments were conducted with the addition of MilliQ H2O only.
Result Leakage occurred rapidly with >50% of the total fluorescence intensity observed after one minute followed by a gradual increase for up to 10 min. The calculated EC50 of kB1 was 61 M compared to 35 M for kB2. These experiments showed that the cyclotides kB1 and kB2 have the capability to alter membrane permeability leading to the loss of the internal vesicle contents to the external medium.
AssayType Membrane Binding Assay

Colgrave ML, Kotze AC, Huang YH, O'Grady J, Simonsen SM, Craik DJ. (2008) Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep. Biochemistry 47:5581-5589

Proteins Assayed kalata B1
kalata B2