Probing the heparin‐binding domain of human antithrombin III with V8 protease
- 1 September 1987
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 167 (2) , 247-252
- https://doi.org/10.1111/j.1432-1033.1987.tb13330.x
Abstract
From structural analysis on genetically abnormal and chemically modified human antithrombin III [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81, 289-293; Chang, J-Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174-1176; Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941], the heparin-binding site of antithrombin III has been suggested to be in the region of Pro-41, Arg-47 and Trp-49. In this study the heparin-binding site was probed by preferential cleavage of V8 protease on heparin-treated and non-treated native antithrombin III. The study has been based on the presumption that the heparin-binding site of antithrombin III is situated at exposed surface domain and may be preferentially attacked during limited proteolytic degestion. Partially digested antithrombin III samples were monitored by quantitative amino-terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1-h-digested antithrombin III was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu-Gly (residues 34-35), Glu-Ala (residues 42-43) and Glu-Leu (residues 50-51) are three preferential cleavages sites for V8 protease and their cleavage, especially the Glu-Ala and Glu-Leu sites, was drastically inhibited when antithrombin III was preincubated with heparin. Both high-affinity and low-affinity antithrombin-III-binding heparins were shown to inhibit the V8 protease digestion of native antithrombin III, but the high-affinity sample exhibited a higher inhibition activity than the low-affinity heparin. These findings (a) imply that the segment containing residues 34-51 is among the most exposed region of native antithrombin III and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.This publication has 55 references indexed in Scilit:
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