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Ammodytoxins (Atxs) are presynaptically neurotoxic secreted phospholipases A2 (sPLA2s) from venom of the long-nosed viper, Vipera ammodytes ammodytes. Ammodytin (Atn) I2 is a non-toxic sPLA2 from the same venom, while AtnL is a myotoxic sPLA2 homologue without enzymatic activity. Although the exact role of enzymatic activity in the process of presynaptic neurotoxicity of sPLA2s isstill unknown, it has been shown that it is essential for full expression ofthe neurotoxic effect. In order to determine the role of different hydrophobic and aromatic residues on the interfacial binding surface (IBS) of Atxs, we prepared a number of recombinant mutant proteins of AtxA. By replacing several residues in the active site and calcium-binding loop of AtnL, we successfully prepared two enzymatically active mutants, which differed only in the substitution V31 W. By carefully analysing the enzymatic characteristics of 16 wild-type and mutant snake venom sPLA2s of group IIA (Atxs, Atns and a weakly neurotoxic sPLA2 from the Russellćs viper), we searched for possible differences in the enzymatic action of neurotoxic and non-neurotoxic sPLA2s. In this study, we show that Atxs are very efficient enzymes when acting on anionic as well as on zwitterionic aggregated phospholipid substrates. Anionic phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles are very good general . substrates for Atxs. The specific enzymatic activities of our snake venom sPLA2s on vesicles composed of neutral phosphatidylcholine (PC) molecules were up to five orders of magnitude lower than that determined on anionic vesicles. The range of activities of the 16 sPLA2s on anionic PG and PS vesicles varied by up to 11- and 34-fold, respectively, while the activities on neutral PC vesicles showed a much higher variability and differed by up to 20,000-fold. (Abstract truncated at 2000 characters).
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Phospholipases A2 (PLA2s) hydrolize fatty acyl ester linkage at the sb-2 position in a phospholipid molecule. They can be divided into high and low molecular mass PLA2s. The low-molecular mass PLA2s are a group of structurallyvery similar proteins, which are usually secretory. However, they are involved in a number of different psychological processes, and their function is not always directly related to their enzymatic activity. Both mammalian tissue and venoms of different snakes are rich sources of low-molecular mass PLA2s. Ammodytoxin C (AtxC) is one of the presynaptically neurotoxic PLA2s found in the venom of the long-nosed viper. It blocks the release of acetylcholine from nerve terminals. In order for AtxC to trigger its neurotoxic effect, it must bind to specific receptor proteins on the target cells. Two high-affinity AtxC-binding proteins, R180 and R25, were recently identified in porcine brain cortex. R180 binds both toxic and non-toxic PLA2s, while R25 is specific for ammodytoxins. We hope that the characterization of these receptor proteins will help us explain the moelcularmechanisms of action of neurotoxic PLA2s. On the other hand, this research should contribute to our understanding of endogenous mammalian PLA2s,which participate in cellular processes also as ligands for specific receptors. We investigated the subcellular localization of R180 and R25 in porcine brain cortex. Both receptors are integral membrane proteins but they do not reside in the same cellular compartments. While R180 is localized on the plasma membranes, R25 is an intracellular protein. Our results suggest that it resides in mitochondria, however, we cannot exclude other cellular structured of similar density. From porcine brain cortex R180 was purified to homogenicity by means of lectin- and AtxC-affinity chromatographies. (Abstracttruncated at 2000 characters).
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