Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen.
We have isolated a serine protease, halystase, from Agkistrodon halys blomhoffii venom by chromatography on DEAE-Sepharose, heparin-Sepharose and Q-Sepharose columns, and have determined the complete amino acid sequence by Edman degradation and by mass spectral analysis of peptides generated by enzymatic and chemical cleavage. The 238-residue sequence of halystase, containing N-linked carbohydrates (about 13%) at two sites showed significant similarity to other thrombin-like snake venom serine proteases (66-72%), mammalian tissue kallikrein (42%) and thrombin (26%). Halystase contained the tentative catalytic triad of His43, Asp88 and Ser184 common to all serine proteases and Asp178 in the primary substrate-binding site. Although halystase contained an RGD sequence at residues 181-183, it did not inhibit platelet aggregation induced by ADP or collagen. It hydrolyzed most efficiently a tissue-kallikrein substrate, prolylphenylalanylarginyl-4-methyl-coumaryl-7-amide, and released bradykinin from bovine kininogen. Halystase did not coagulate human plasma, but it cleaved the fibrinogen B beta chain at the carboxyl side of Arg42 and cleaved slowly the fibrogen A alpha chain. Fibrinogen thus treated gradually became insensitive to thrombin. The proteolytic activity was inhibited with diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride or leupeptin. These results indicate that halystase is a serine protease structurally similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves fibrinogen at sites different from thrombin without inducing fibrin clotting, and hydrolyzes kininogen to produce bradykinin, resulting in the reduction of blood pressure.
Matsui
T
T
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.
Sakurai
Y
Y
Fujimura
Y
Y
Hayashi
I
I
Oh-Ishi
S
S
Suzuki
M
M
Hamako
J
J
Yamamoto
Y
Y
Yamazaki
J
J
Kinoshita
M
M
Titani
K
K
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000201:enzymology*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007704:Kininogens / Q000378:metabolism; D007705:Kinins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018629:Spectrometry, Mass, Secondary Ion; D013379:Substrate Specificity
D003435:Crotalid Venoms; D007704:Kininogens; D007705:Kinins; D010446:Peptide Fragments; D005340:Fibrinogen; D012697:Serine Endopeptidases; C111796:halystase