ABSTRACT
Three isoenzymes of phospholipase A2 (PLA2), VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX were isolated from Daboia russelii snake venom. The venom, upon gel filtration on Sephadex G-75 column, resolved into six peaks (DRG75 I-VI). The VRV-PL-IIIc was purified by subjecting DRG75II to homogeneity by rechromatography in the presence of 8M urea on Sephadex G-75 column. The other two isoenzymes VRV-PL-VII and VRV-PL-IX were purified by subjecting DRG75III to ion exchange chromatography on CM-Sephadex C-25 column. Mol wt. for the three PLA2s, VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX are 13.003kDa, 13.100kDa and 12.531kDa respectively. The VRV-PL-IIIc is not lethal to mice up to 14mg/kg body weight but it affects blood sinusoids and causes necrosis of the hepatocytes in liver. It causes hemorrhage in kidney and shrinkage of renal corpuscles and renal tubules. The LD50s for VRV-PL-VII and VRV-PL-IX are 7 and 7.5mg/kg body weight respectively. They induced neurotoxic symptoms similar to VRV-PL-V. All the three PLA2s are anticoagulant and induced varying degree of edema in the foot pads of mice. VRV-PL-V and VRV-PL-VII are shown to act as pre and post synaptic toxins, while VRV-PL-IX acts as presynaptic toxin. This is evident from experiments conducted on cultured hippocampal neurons by patch clamp electrophysiology.
Subject(s)
Phospholipases A2/chemistry , Phospholipases A2/pharmacology , Snake Venoms/chemistry , Viper Venoms/chemistry , Animals , Anticoagulants/adverse effects , Anticoagulants/chemistry , Anticoagulants/pharmacology , Edema/chemically induced , Hemorrhage/chemically induced , Hepatocytes/drug effects , Isoenzymes/chemistry , Isoenzymes/pharmacology , Kidney Tubules/drug effects , Liver/drug effects , Male , Mice , Necrosis/chemically induced , Phospholipases A2/adverse effectsABSTRACT
Bile acids, such as cholic acid (CA) and ursodeoxycholic acid (UDCA) have shown to decrease or increase the enzymatic activity of group IB pancreatic PLA(2), depending on the concentration used. Studies suggest that the inhibition of hydrolysis rate of the substrate is due to formation in aqueous phase of a complex between bile acid and PLA(2), which is catalytically inert. For this reason, we tested the inhibition of the enzymatic activity of group IIA snake venom PLA(2) by bile acids, using an aqueous phase model. In addition, we measured the ability of bile acids to inhibit the toxic effects caused by the mentioned toxin. UDCA and CA inhibited the enzymatic activity of the PLA(2) in a competitive mode. Moreover, these compounds inhibited myotoxic, cytotoxic and edema-forming activities induced by the toxin, but UDCA was more efficient than CA. It was demonstrated that bile acids interact directly with this protein by causing slight changes in the intrinsic fluorescence spectra. Preliminary molecular docking studies suggest that bile acids interact with amino acids at the active site of the PLA(2) through different interactions, CA showed hydrogen bonds with His48, whereas, UDCA displayed with Asp49. Results obtained herein may turn UDCA and CA into promising models for the development of new molecules with anti-inflammatory and anti-snake venom PLA(2) properties.
