Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A2 Noradrenergic Lesions Prevent Renal Sympathoinhibition Induced by Hypernatremia in Rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desnaturação de oxihemoglobinas humanas A, A2 e S pelo metodo de tampão isopropanol

Human oxyhaemoglobin A and A2 from normal individuals and oxyhaemoglobin S from patients with sickle cell anaemia and sickle cell trait were studied using Isopropanol/buffer method at 37°C and 40°C. Hb S was less stable than Hb A, whereas Hb A2 was considerably more stable than either. Denaturation of Hb S was dependent on temperature and its concentration. Between the patients with sickle cell trait it was not possible to verify the influence of the concentration probably due to the small range used (from 38% to 44%).

Polybitoxins: A group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista)

The neotropical wasp Polybia paulista is very aggressive and endemic in south-east Brazil, where it frequently causes stinging accidents. By using gel filtration on Sephadex G-200, followed by ion-exchange chromatography on DEAE-Cellulose under a pH gradient, a group of four toxins (designated as polybitoxins-I, II, lII and IV) presenting phospholipase A2 (PLA2) activities was purified. These toxins are dimeric with mol. wts ranging from 115,000 to 132,000 and formed by different subunits. The four toxins contain very high sugar contents attached to their molecules (22-43% w/w) and presented different values of pH optimum from 7.8 to 9.0; when dissociated, only residual catalytic activities were maintained. The catalytic activities of polybitoxins (from 18 to 771 μmoles/mg per minute) are lower than that of PLA2 from Apis mellifera venom and hornetin from Vespa basalis. The polybitoxins presented a non-linear steady-state kinetic behavior for the hydrolysis of phosphatidylcholine at pH 7.9, compatible with the negative co- operativity phenomena. All of the polybitoxins were very potent direct hemolysins, especially the polybitoxins-III and IV, which are as potent as the lethal toxin from V. basalis and hornetin from Vespa flavitarsus, respectively; polybitoxin-IV presented hemolytic action 20 times higher than that of PLA2 from A. mellifera, 17 times higher than that of neutral PLA2 from Naja nigricolis and about 37 times higher than that of cardiotoxin from Naja naja atra venom.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Crystallization and preliminary X-ray analysis of SIII-SPIII, a ahospholipase A2 isolated from the venom of Bothrops jararacussu

Large single crystals have been obtained of SIII-SPIII, a phospholipase A2 from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Å. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.

A theoretical study of the A2 ∑+-X2∏ system of the SiP molecule

The A2∑+ and Z2∏ electronic states of the SiP species have been investigated theoretically at a very high level of correlation treatment (CASSCF/MRSDCI). Very accurate potential energy curves are presented for both states, as well as the associated spectroscopic constants as derived from the vib-rotational energy levels determined by means of the numerical solution of the radial Schrödinger equation. Electronic transition moment function, oscillator strengths, Einstein coefficients for spontaneous emission, and Franck-Condon factors for the A2∑+-X2∏ system have been calculated. Dipole moment functions and radiative lifetimes for both states have also been determined. Spin-orbit coupling constants are also reported. The radiative lifetimes for the A2∑+ state, taking into account the spin-orbit diagonal correction to the X2∏ state, decrease from a value of 138 ms at v′ = 0 to 0.48 ms at v′ = 8, and, for the X2∏ state, from 2.32 s at v″ = 1 to 0.59 s at v″ = 5. Vibrational and rotational transitions are expected to be relatively strong.

Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom

Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 Å resolution and the anion-binding site has been characterized. © 2006 International Union of Crystallography. All rights reserved.

Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. © 2008 Elsevier Inc. All rights reserved.

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A2 (PLA2) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA2 homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA2 homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA2 homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement. © 2012 Elsevier B.V.