Zinc and calcium ions cooperatively modulate ADAMTS13 activity
Zinc and calcium ions cooperatively modulate ADAMTS13 activity
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DOI：
10.1074/jbc.m504540200
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发表时间：
2006-01-13
影响因子：
4.8
通讯作者：
Sadler, JE
中科院分区：
文献类型：
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作者：
Anderson, PJ;Kokame, K;Sadler, JE
ADAMTS 13是一种切割血管性血友病因子（VWF）多聚体的金属蛋白酶。用多聚体VWF和基于VWF A2结构域的Asp（1596）-Arg（1668）的荧光肽底物FRETS-VWF 73检查ADAMTS 13活性的金属离子依赖性。柠檬酸盐抗凝血浆中的ADAMTS 13活性被锌离子增强了2倍，被钙离子增强了3倍，被两种离子增强了6倍，表明协同激活。重组ADAMTS 13对VWF的切割被锌离子（K-D（app）类似于0.5，类似于M）、钙离子（K-D（app）类似于4.8，类似于M）和钡离子（K-D（app）类似于1.7 mM）激活高达200倍。钡离子刺激ADAMTS 13活性在柠檬酸盐血浆中，但不是在无柠檬酸盐血浆。因此，柠檬酸盐血浆中ADAMTS 13的钡离子刺激似乎反映了螯合钙和锌离子从柠檬酸盐复合物中的释放。在最佳锌和钙浓度下，ADAMTS 13以3.7 +/- 1.4 μ/ml的Km app（类似于VWF亚基的15 nM）切割VWF，这与5-10 μ g/ml的血浆VWF浓度相当。ADAMTS 13可以切割类似于14%的盐酸胍预处理的VWF，这表明该底物对蛋白水解的敏感性是异质的。ADAMTS 13以3.2 +/- 1.1 μ M的K-m（app）切割FRETS-VWF 73，与VWF相比亲和力降低200倍一致。ADAMTS 13裂解VWF和FRETS-VWF 73的催化效率大致相当，分别为55 μ M-1 min（-1）和18 μ M-1 min（-1）。ADAMTS 13对VWF的显著偏好表明底物识别依赖于FRETS-VWF 73中缺失的多聚体VWF上的结构特征或外位点。
ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced similar to 2-fold by zinc ions, similar to 3-fold by calcium ions, and similar to 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to similar to 200-fold by zinc ions (K-D (app) similar to 0.5 similar to M), calcium ions (K-D (app) similar to 4.8 similar to M), and barium ions (K-D (app) similar to 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a Km app of 3.7 +/- 1.4 mu/ml (similar to 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 mu g/ml. ADAMTS13 could cleave similar to 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K-m (app) of 3.2 +/- 1.1 mu M, consistent with an 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 mu M-1 min(-1) and 18 mu M-1 min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.