Role of calcium(II) ions in the recognition of coagulation factors IX and X by IX/X-bp, an anticoagulant from snake venom.
IX/X-bp, an anticoagulant protein isolated from the venom of the habu snake Trimeresurus flavoviridis, has a structure homologous to the carbohydrate-recognition domains of C-type (Ca(2+)-dependent) animal lectins, and it binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX and X in a Ca(2+)-dependent fashion. In the present study, we elucidated the role of Ca2+ ions in this binding. The binding of 125I-labeled IX/X-bp to both coagulation factors required about 1 mM Ca2+ ions in this at pH 7.5. A decrease in the pH to 6.5 had a striking negative effect on the binding, and the Ca(2+)-requirement curve was shifted rightward. We investigated the binding of Ca2+ ions to IX/X-bp directly by equilibrium dialysis and identified two independent binding sites with different affinities. At pH 7.5, the apparent Kd values for these sites were 25 and 200 microM, respectively. When the pH was decreased to 6.5, the affinity of the high-affinity binding site was reduced only slightly but that of the low-affinity site was reduced considerably. Moreover, it was evident from observations of Ca(2+)-induced changes in the intrinsic fluorescence that IX/X-bp underwent a conformational change upon binding of Ca2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Sekiya
F
F
Department of Biochemistry, Meiji College of Pharmacy, Tokyo, Japan.
Yamashita
T
T
Morita
T
T
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001779:Blood Coagulation Factors / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003435:Crotalid Venoms / Q000378:metabolism*; D005164:Factor IX / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D030162:Reptilian Proteins; D013050:Spectrometry, Fluorescence; D017841:Trimeresurus
D000925:Anticoagulants; D001779:Blood Coagulation Factors; D002352:Carrier Proteins; D003435:Crotalid Venoms; D030162:Reptilian Proteins; C070302:factor IX-factor X-binding protein, Crotalinae; D005164:Factor IX; D005170:Factor X; D002118:Calcium