Avtor/Urednik		Ivanovski, Gabriela
Naslov		Študij encimskega in toksičnega delovanja amoditoksinov
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		2001
Obseg		str. 93
Jezik		slo
Abstrakt		The Vipera ammodytes ammodytes venom contains three presynaptically neurotoxic PLA2S, ammodytoxins (atx) A, B and C, of which atxA is the most lethal. The molecular mechanism of presynaptic toxicity and the amino acid residues involved in toxicity are not known. Site-directed mutagenesis was used to determine whether the basic amino acid residues in the B-structure of atxA are involved in the process of neurotoxicity. Using polymerase chain reaction, we prepared nine mutants of atxA: R72S, R72I, R72K, R72E, K86A, K86E, K86G, K86R and K74S/H76S/R77L. We examined the effect of negatively charged, polar uncharged and nonpolar amino acid residues on the enzymatic activity and toxicity of atxA. Also we examined the effect of exchanging one basic amino acid residue by another. All the mutants were produced as fusion proteins in the Escherichia coli expression system. The specific enzymatic activity of the R72I, K86E and K74S/H76S/R77L mutants increased, whereas that of the R72E and R72K mutants decreased to natural atxA. In the case of R72S, K86A, K86G and K86R mutants there was no significant influence on the specific enzymatic activity. We propose that charge redistribution and increased hydrophobicity on the surface of atxA have a positive effect on the binding of the PLA2 molecule to the lipid-water interface and thus on the enzymatic activity. The binding affinities of all mutant proteins for the specific neuronal receptors from porcine brain (R180, R25 and colmodulin) were only slightly less than that of native atxA, which is in accord with the small change in toxicity of all the mutants. These results indicate that basic amino acid residues in the B-structure are important, but not critical, for the neurotoxic effect of ammodytoxins. (Abstract trunacted at 2000 characters).
Deskriptorji		VIPER VENOMS PHOSPHOLIPASES A RECOMBINANT FUSION PROTEINS ESCHERICHIA COLI PICHIA PLASMIDS CLONING, MOLECULAR POLYMERASE CHAIN REACTION BASE SEQUENCE TRANSFORMATION, GENETIC VIPERIDAE