Different effects of nine clausenamide ennatiomers on liver glutathione biosynthesis and glutathione S-transferase activity in mice.

AIM: To study the effects of nine synthetic clausenamide with different stereo structures on liver glutathione (GSH) biosynthesis and glutathione S-transferase (GST) activity in mice. METHODS: The nine test compounds were racemic mixtures and their ennatiomers of clausenamide, neoclausenamide and epineoclausenamide. Mice were administered clausenamide 250 mg/kg once daily for 3 consecutive days, ig, and were killed 24 h after the last dosing. The mouse liver cytosol GSH and GST were determined with related biochemical methods. RESULTS: Nine clausenamides exhibited different effects on liver GSH and GST. Of nine clausenamides, only (+) and (+/-)clausenamide markedly increased liver cytosol GSH content. The mechanism of increasing liver GSH content of (+)clausenamide is mainly due to stimulating the key limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS) activity for GSH biosynthesis. The other test clausenamides had no such effect on liver GSH. All of the nine clausenamides induced a significant increase of GST activity. CONCLUSION: The effects of clausenamide ennatiomers on liver GST and GSH varied with the alterations of their spatial structures. (+)Clausenamide stimulated liver GSH biosynthesis through enhancing gamma-GCS activity.

Shedaoenase, a novel fibrinogenase from the venom of Agkistrodon shedaoenthesis Zhao.

Shedaoenase, a serine protease, was isolated from the venom of Agkistrodon shedaoenthesis Zhao with an apparent molecular mass of 36 kDa. It was purified by affinity chromatography on arginine Sepharose 4B column and anion exchange on Mono Q fast protein liquid chromatography. Shedaoenase preferentially cleaved the Aalpha-chain of human fibrinogen and slowly digested the Bbeta-chain. It also showed arginyl esterase activity using Nalpha-benzoyl-L-arginine ethyl ester as a substrate, and some synthetic chromogentic substrates, such as Chromozym PL, S-2266, and S-2160, could also be hydrolyzed. The enzyme activity of shedaoenase could be completely inhibited by phenylmethylsulphonylfluoride and could be little inhibited by the chelating reagent EDTA. The N-terminal sequence of shedaoenase was determined, and its full-length cDNA encoding a protein of 238 amino acid residues was cloned by reverse transcription-polymerase chain reaction from the total mRNA extracted from the snake venom gland. The deduced primary sequence of shedaoenase shares significant homology with other snake venom serine proteases.

Purification, gene cloning and expression of an acidic phospholipase A2 from Agkistrodon shedaoensis Zhao.

A protein with the activity of phospholipase A(2) named asAPLA(2) was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S, and Mono Q FPLC. Its molecular weight was estimated to be 19 kD by SDS-PAGE, and its pI was about 3.5 by IEF analysis. It inhibited the platelet aggregation that was induced by 1 micromol/ L ADP, and the IC(50) was determined to be 6 micromol/L. Degenerating primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA(2). Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. Its molecular weight and the pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar software according to the deduced amino acid sequence. Then the gene was cloned into the expression plasmid pET-40b(+) and expressed in E. coli BL21(DE3). Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme.