Rhodocetin, a Novel Platelet Aggregation Inhibitor from the Venom of Calloselasma rhodostoma (Malayan Pit Viper): Synergistic and Noncovalent Interaction between Its Subunits,
- 22 May 1999
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 38 (23) , 7584-7593
- https://doi.org/10.1021/bi982132z
Abstract
A novel platelet aggregation inhibitor, rhodocetin, was purified from the crude venom of Calloselasma rhodostoma. It inhibited collagen-induced platelet aggregation in a dose-dependent manner, with an IC50 of 41 nM. Rhodocetin has a heterodimeric structure with α and β subunits, which could be separated on a nonreducing denaturing gel or reverse-phase HPLC column. Individually neither subunit inhibited platelet aggregation even at 2.0 μM concentration. Titration and reconstitution experiments showed that, when these subunits are mixed to give a 1:1 complex, most of its biological activity was recovered. The reconstituted complex inhibited platelet aggregation with an IC50 of 112 nM, about 3-fold less effective than the native molecule. Circular dichroism analysis revealed that the reconstituted complex had a spectrum similar to that of the native protein. By using surface plasmon resonance studies, we established that the stoichiometry of binding between the two subunits is 1:1 and the subunits interact with a Kd of 0.14 ± 0.04 μM. The complete amino acid sequences of the α (15956.16 Da, 133 residues) and β (15185.10 Da, 129 residues) subunits show a high degree of homology with each other (49%) and with the Ca2+-dependent lectin-related proteins (CLPs) (typically 29−48%) isolated from other snake venoms. Unlike the other members of the family in which the subunits are held together by an interchain disulfide bond, rhodocetin subunits are held together only through noncovalent interactions. The cysteinyl residues forming the intersubunit disulfide bridge in all other known CLPs are replaced by Ser-79 and Arg-75 in the α and β subunits of rhodocetin, respectively. These studies support the noncovalent and synergistic interactions between the two subunits of rhodocetin. This is the first reported CLP dimer with such a novel heterodimeric structure.Keywords
This publication has 8 references indexed in Scilit:
- Amino acid sequence of the α subunit and computer modelling of the α and β subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)Biochemical Journal, 1997
- Complete Amino Acid Sequence and Identification of the Platelet Glycoprotein Ib-binding Site of Jararaca GPIb-BP, a Snake Venom Protein Isolated from Bothrops jararacaJournal of Biological Chemistry, 1996
- Blood Coagulation Factor IX-Binding Protein from the Venom of Trimeresurus flavoviridis: Purification and CharacterizationThe Journal of Biochemistry, 1995
- Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregationBiochemical Journal, 1995
- Isolation, Characterization and Amino Acid Sequence of Echicetin β Subunit, a Specific Inhibitor of von Willebrand Factor and Thrombin Interaction with Glycoprotein IbBiochemical and Biophysical Research Communications, 1994
- Refolding proteins by gel filtration chromatographyFEBS Letters, 1994
- The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin EJournal of Biological Chemistry, 1991
- Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.Journal of Biological Chemistry, 1990