DOMAIN-STRUCTURE OF CALPAIN - MAPPING THE BINDING-SITE FOR CALPASTATIN
DOMAIN-STRUCTURE OF CALPAIN - MAPPING THE BINDING-SITE FOR CALPASTATIN
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DOI：
10.1021/bi00249a008
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发表时间：
1994-11-15
期刊：
影响因子：
2.9
通讯作者：
MCGRODY, KS
中科院分区：
文献类型：
--
作者：
CROALL, DE;MCGRODY, KS
合成肽EKLGERDDTIPPEYALEKKTGV以模拟钙蛋白酶抑制素的中心共有序列，钙蛋白酶抑制素是钙蛋白酶的特异性内源性抑制剂（EC 3.4.22.17）。该肽竞争性抑制酪蛋白被微钙蛋白酶或毫钙蛋白酶水解，但不影响其他蛋白酶的活性。这种抑制肽优先交联到毫钙蛋白酶在钙的存在下，使用异双功能交联剂m-马来酰亚胺苯甲酰基-N-羟基琥珀酰亚胺酯。肽的交联被钙蛋白酶抑制素阻断。使用酶-肽复合物在半胱氨酸残基处的随机化学裂解，定位在毫钙蛋白酶内肽的交联位点。钙蛋白酶片段通过与肽特异性抗血清的反应性鉴定为氨基末端片段，或通过掺入来自（CN）-C-14的C-14鉴定为非氨基末端片段。从使用毫钙蛋白酶和微钙蛋白酶的实验中分析对照和交联片段，将化学交联位点映射到半胱氨酸-497，并将钙蛋白酶抑制剂样肽的结合位点定位到钙蛋白酶催化亚基的结构域III的高度保守区域。
The peptide EKLGERDDTIPPEYRELLEKKTGV was synthesized to mimic the central consensus sequence of calpastatin, the specific, endogenous inhibitor of the calpains (EC 3.4.22.17). The peptide competitively inhibits hydrolysis of casein by either micro- or milli-calpain but does not affect the activity of other proteases. This inhibitory peptide was preferentially cross-linked to milli-calpain in the presence of calcium using the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Cross-linking of the peptide was blocked by calpastatin. The site of crosslinking for the peptide within milli-calpain was localized using random chemical cleavage of the enzyme-peptide complex at cysteine residues. Calpain fragments were identified as amino-terminal fragments through reactivity with a peptide-specific antiserum or as non-amino-terminal fragments through incorporation of C-14 from (CN)-C-14. Analysis of the control and cross-linked fragments, from experiments using both milli-calpain and micro-calpain, maps the chemical cross-linking site to cysteine-497 and localizes the binding site for the calpastatin-like peptide to this highly conserved region of domain III of calpains catalytic subunit.