RESUMO
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.
Assuntos
Agkistrodon/metabolismo , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Reações Cruzadas , Fibrinogênio/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/imunologia , Especificidade por SubstratoRESUMO
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.
Assuntos
Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Fosfolipases A/química , Fosfolipases A/metabolismo , Análise de Sequência de Proteína , Agkistrodon/genética , Agkistrodon/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ativação Enzimática , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Isoenzimas , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/imunologia , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Homologia de Sequência de AminoácidosRESUMO
To identify the anticoagulant region of the phospholipase A(2) (PLA(2)) from the Agkistrodon halys Pallas (class II), four mutants E53G, W70M, T56K, and D67K were produced according to the prediction from the crystal structure and the sequence comparison of the strong, weak and non-anticoagulant PLA2s. A test of blood clotting revealed that E53G and W70M had lost their effects on the blood clotting, while T56K and D67K had enhanced activity. The four residues are located on the same face in the tertiary structure of this enzyme. The result supported the prediction that there exists an anticoagulant region that is composed of some residues that are close to each other in tertiary structure to form a functional face.
