ABSTRACT
Aim To purify L-amino acid oxidase(LAAO) from the venom of Naja atra and study its effect on endothelial cells.Methods The NAV-LAAO was purified by ion-exchange chromatography and affinity chromatography.The MTT assay and Western blot were used to detect the viability and apoptosis of HUVEC.The tubule-forming was used to study the angiogenesis of cells.Results The NAV-LAAO was purified successfully from the venom of Naja atra.The molecular weight of NAV-LAAO was determined to be 58 ku by SDS-PAGE.NAV-LAAO effectively inhibited the growth and tubule-forming of HUVEC,and the 50% inhibitory concentration(IC50) was 21.42 mg?L-1.Compared with control,the levels of caspase-3 and caspase-8 increased in HUVEC treated with NAV-LAAO.Conclusions The NAV-LAAO is purified successfully from Naja atra venom by ion-exchange chromatography and affinity chromatography.The NAV-LAAO inhibits the growth and tubule-forming capacity of HUVEC in a dose-dependent manner.
ABSTRACT
AIM To purify cardiotoxin from Naja atra venom and investigate the relationship between cardiotoxicity of cardiotoxin and coronary artery spasm induced by cardiotoxin. METHODS Cardio toxin 13 (CTX 13) was fractionated and purified by chromatography and gel filtration from Chinese cobra (Naja atra) venom. The cardiotoxicity were observed in rat in situ, its isolated heart preparation and papillary muscle preparations. RESULTS Ion exchange chromatography of lyophilized cobra venom on SP Sephadex C 50 yielded 15 fractions, of thses fractions, cardiotoxic activities were found in fraction 11, 12, 13, and 14. Gel filtration and Ion chromatography of fraction 13 on Sephadex G 50 and SP Sephadex C 25 were performed consecutively and CTX 13 was obtained. It was homogeneous on polyacrylamide gel electrophoresis with MW= 7 769 ku, and 60 amino acid residues. The iv LD 50 in mice was 0 756 mg?kg -1 . CTX 13 increased the coronary resistance and reduced the contractility of rat Langendorff heart preparations. Systolic standstill finally occurred. When the heart preparations were pretreated with nitrendipine, an calcium channel blocker, the resistance seldom increased. The contractility slightly decreased at the beginning and then significantly increased. The tonus of contraction did not occurred. CTX 13 induced dose dependent contraction of pig coronary artery ring segments. Nitrendipine inhibited the action of CTX 13 on the coronary ring segments. However, nitrendipine had no effects on the action of CTX 13 in the rat papillary muscle preparations. The MLD of CTX 13 by venoclysis was changed from (444 7?28 5) ?g?kg -1 to (541 1?23 2) ?g?kg -1 in anaesthetized rats while the rats were pretreated with nitrendipine. CONCLUTION The coronary artery spasm may be one of the causes of death due to CTX 13.