Browsing by Subject "Gift"

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Isolierung und funktionelle Charakterisierung von Einzelkomponenten des Gifts der südamerikanischen Klapperschlange Crotalus durissus terrificus
(2017) Kaltenbach, Stefan; Hanke, Wolfgang
In order to understand the pharmacological activities of proteins and peptides it is important to study the relation of their molecular structure and their mode of action. Snake venoms contain a large number of biologically active compounds. They influence and damage the organism of prey in various manners. Therefore, venoms are an abundant source of substances with biological activity. The isolation of these substances is an important way to search for new, pharmacologically active proteins and peptides. The presented work focused on the isolation and characterization of venom compounds from the South American rattlesnake Crotalus durissus terrificus. Two of its main compounds, crotamine and a mixture of Crotoxin B isoforms, were isolated by RP-HPLC and identified by mass spectrometry. Afterwards, the emphasis was laid on how crotamine interacts with artificial membranes. Crotamine is a small, amphipathic and highly basic polypeptide with a molecular weight of 4.9 kDa and a high positive surface charge. It used to be described as a neurotoxin, but meanwhile many additional properties have been reported. These include myotoxic, analgetic, antimicrobial and antitumor activities. To some extent, these modes of action are based on the interaction of crotamine with the membrane lipids. In this context, the membrane modifying properties of crotamine and its ability to translocate into cells are under discussion. The presented work clearly demonstrates that the lipid composition of membranes influences the membrane modifying properties of crotamine. In lipid monolayers built from asolectin, cholesterol had the effect, that crotamine is integrated into the membrane more slowly. Furthermore, it is obvious that the presence of bivalent cations as well as an increasing lateral pressure inside the monolayer leads to a faster integration of crotamine. Moreover, crotamine altered the membrane fluidity of vesicles. In pure asolectin-vesicles crotamine stiffened the membranes (decreased fluidity), whereas it increased the fluidity in DOPC-vesicles. In vesicles containing a mixture of either asolectin or DOPC and cholesterol, crotamine increased the membrane fluidity, whereby its influence was weakened with rising concentrations of cholesterol. However, the impact of cholesterol was stronger in asolectin:cholesterol-vesicles. Hence, cholesterol reduces the membrane modifying properties of crotamine. It was assumed that this is due to the fact that cholesterol decrease the fluidity of membranes. Also, cholesterol may reduce the possibility of polar lipid head groups interacting with cationic molecules like crotamine. As asolectin contains negatively charged lipids, it was concluded that these lipids could be responsible for the stronger impact crotamine had on the fluidity of membranes. They could lead to a stronger attraction of crotamine, due to the more negatively charged membrane surface. Negatively charged lipids might also increase the possibility of raft building, caused by crotamine. In conclusion cholesterol as well as negatively charged lipids alter the effect crotamine has on membranes. It is also influenced by bivalent cations in the surrounding solution as well as the lateral pressure of the membrane. In addition, it was shown that crotamine also affects the calcium homeostasis of neuronal cells. By enlarging the amount of crotamine the internal free calcium concentration also showed an increase.