Characterization of a thrombin-like serine protease, Kangshuanmei, isolated from the venom of a Chinese snake, Agkistrodon halys brevicaudus stejneger.
An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.
Zhang
S
S
Department of Physiology, School of Medicine, Aichi Medical University, Nagakute, Japan.
Ma
B
B
Sakai
J
J
Shiono
H
H
Matsui
T
T
Sugie
I
I
Okada
T
T
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects*; D003435:Crotalid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006490:Hemostatics / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D013917:Thrombin / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology
D003435:Crotalid Venoms; D006490:Hemostatics; D005340:Fibrinogen; D012697:Serine Endopeptidases; C441966:kangshuanmei; D013917:Thrombin