Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix

Biochemistry ◽  
1988 ◽  
Vol 27 (7) ◽  
pp. 2558-2564 ◽  
Author(s):  
Carolyn L. Orthner ◽  
Prabir Bhattacharya ◽  
Dudley K. Strickland
Keyword(s):  
Protein C ◽  
Agkistrodon Contortrix ◽  
Agkistrodon Contortrix Contortrix

PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX

1987 ◽  
Author(s):  
Carolyn L Orthner ◽  
Prabir Bhattacharya ◽  
Dudley K Strikland
Keyword(s):  
Protein C ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Agkistrodon Contortrix ◽  
Purification And Properties ◽  
Sds Page ◽  
Single Protein ◽  
Agkistrodon Contortrix Contortrix

There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.

scholarly journals Characterization of a protein C activator from Agkistrodon contortrix contortrix venom.

1987 ◽  
Vol 262 (26) ◽  
pp. 12607-12613 ◽  
Author(s):  
W Kisiel ◽  
S Kondo ◽  
K J Smith ◽  
B A McMullen ◽  
L F Smith
Keyword(s):  
Protein C ◽  
Agkistrodon Contortrix ◽  
Agkistrodon Contortrix Contortrix

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 674-679 ◽  
Author(s):  
Brad A. McMullen ◽  
Kazuo Fujikawa ◽  
Walter Kisiel
Keyword(s):  
Primary Structure ◽  
Protein C ◽  
Agkistrodon Contortrix ◽  
Agkistrodon Contortrix Contortrix
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