A novel phospholipase A2 (D49) from the venom of the Crotalus oreganus abyssus (North American Grand canyon rattlesnake).

Currently, Crotalus viridis was divided into two species: Crotalus viridis and Crotalus oreganus. The current classification divides "the old" Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concolor, oreganus, helleri, cerberus, and caliginis). The analysis of a product from cDNA (E6d), derived from the gland of a specie Crotalus viridis viridis, was found to produce an acid phospholipase A2. In this study we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA2 from E6d cDNA is actually the same PLA2 presented in the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data.

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom.

Crotalus oreganus abyssus is a rattlesnake that is usually found in the Grand Canyon, United States of America. Knowledge regarding the composition of C. o. abyssus venom is scarce. New natriuretic peptides (NPs) have been isolated and characterized from the venoms of members of the Crotalinae family. The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. The aim of the present study was to characterize a novel natriuretic-like peptide (Coa_NP2), isolated from C. o. abyssus venom. The Coa_NP2 presents an average molecular mass of 3419.88Da (theoretical average molecular mass 3418.94Da, monoisotopic molecular mass 3416.66Da and theoretical PI 7.78) and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. The peptide has 32 amino acids and its complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. Coa_NP2 is a natriuretic peptide of the ANP/BNP-like family, since the carboxyterminal region of CNP has its own NP domain. We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations of markers of nitric oxide formation measured in the plasma and vasorelaxation in a thoracic aortic ring bath. The structural and biological aspects confirm Coa_NP2 as a new natriuretic peptide, isolated from snake venom.

Molecular modeling and inhibition of phospholipase A2 by polyhydroxy phenolic compounds.

Phospholipases A(2) are enzymes responsible for the hydrolysis of membrane phospholipids that release arachidonic acid, which serves as substrate for pro-inflammatory mediators, such as prostaglandins and leucotriens. The design of specific inhibitors for PLA(2) might help in the development of new anti-inflammatory drugs. Polyhydroxy phenolic compounds, such as flavonoids, vitamin E, rosmarinic acid and aristolochic acid, are able to inhibit PLA(2) from different sources. Herein, we have studied the kinetic behavior and the capacity of inhibiting edema formation induced by PLA(2) of five different polyhydroxy phenolic compounds (two phenolic derivatives and three acetophenone hydroxylated derivatives) extracted from the venom of Crotalus adamanteus. The results showed that compounds 1,3-dihydroxy benzene, 1,3,5-trihydroxy benzene and 2,4,6-trihydroxy acetophenone were the most efficient in the inhibition of the enzymatic activity and edema induction by PLA(2). It was also verified that the number of hydroxyls in each molecule is not a limiting factor for the inhibition capacity of these compounds. Molecular modeling studies indicated that the most active compounds are linked to the amino acid Asp 49 and that they destabilize the coordination of the calcium atom, which is essential to the catalytic activity. The study of potential surfaces showed that there are conditions in which the potential values must be adequate for enzyme complex formation with polyhydroxy phenolic compounds. When the potential over the hydroxyl surfaces is very high, formation of stable complexes does not occur and the enzyme does not act intensely. These results might be helpful in the design of a drug that specifically inhibits PLA(2).

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma.

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.