Fractionation of snake venom metalloproteinases by metal ion affinity: A purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni-agarose |
| |
Authors: | Lakshmi C Wijeyewickrema Elizabeth E Gardiner Yang Shen Michael C Berndt Robert K Andrews |
| |
Institution: | aDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, 3008 Victoria, Australia bDepartment of Immunology, Monash University, Alfred Medical Research and Education Precinct (AMREP), Commercial Road, Melbourne, 3004 Victoria, Australia |
| |
Abstract: | Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni2+-agarose, and was eluted by 10 mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ib , with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIb extracellular fragment (glycocalicin), and inhibition of 125I-VWF binding to GPIb on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni2+-agarose. Samples of flow-through, wash, and imidazole-eluted (0–30 mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni2+-agarose to fractionate snake venom metalloproteinases. |
| |
Keywords: | Glycoprotein Ib–IX–V Glycoprotein VI Metalloproteinases Platelets Snake venoms |
本文献已被 ScienceDirect 等数据库收录! |
|