Effect of bilineobin, a thrombin-like proteinase from the venom of common cantil (Agkistrodon bilineatus).
A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24.
Komori
Y
Y
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
Nikai
T
T
Ohara
A
A
Yagihashi
S
S
Sugihara
H
H
D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity*; D005176:Factor XIII / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000633:toxicity*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D011480:Protease Inhibitors; D005340:Fibrinogen; D005176:Factor XIII; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D010450:Endopeptidases; D012697:Serine Endopeptidases; C080674:bilineobin; D013917:Thrombin