The gram-positive, spore forming bacteria Bacillus thuringiensis synthesize cytoplasmic crystalline inclusions, the Bt proteins, which are toxic to insect larvae. Although Bt proteins have been used as bioinsecticides for many years and a considerable number of studies have been carried out to investigate their mode of action, their precise mechanism of action still remains unclear. A 27 kDa protein from Bt subsp. israelensis (cytA27) is known for its broad cytolytic activity against a wide range of invertebrate and vertebrate cells in vitro as well as mosquitocidal activity in vivo. Given the interest in the mechanism of the membrane interaction of cytA27 and its proteolytic product, a 24 kDa protein (cytA24), we have undertaken a study of their interactions with model phospholipid membranes. The cytA27 and cytA24 proteins were purified by selective solubilization and ion exchange HPLC and characterized by SDS-PAGE, N-terminal sequencing and mass spectrometry. Fluorescence spectroscopy was used to characterize the cytA-induced release of fluorescence markers from vesicles and the cytA-vesicle affinity and stoichiometry. The results indicate that cytA proteins bind to lipids and affect the integrity of phospholipid vesicles. The binding is non-specific in that it does not require a receptor. CytA27 and cy8tA24 interact with PC-LUV with apparent binding constants of $\rm (0.34\pm0.02)\times10\sp5M\sp{-1}$ and $\rm (1.54\pm0.10)\times10\sp5M\sp{-1}$ respectively. Binding isotherm data indicate that a critical number of cytA molecules must associate with the membrane in order to induce vesicle leakage. Approximately 324 of cytA27 and 157 of cytA24 molecules are required to bind to one PC-LUV before the latter starts to release its contents. CytA-induced vesicle leakage follows an all-or-none mechanism, i.e. each LUV either releases all of its contents or remains intact. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/6541 |
| Date | January 1994 |
| Creators | Huang, Fang. |
| Contributors | Carey, Paul, |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 156 p. |
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