All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli.
All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli.
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钙依赖性蛋白酶内源抑制剂的所有四个重复结构域独立地保留抑制活性。
DOI：
10.1016/s0021-9258(18)69215-4
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
K. Suzuki
中科院分区：
文献类型：
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作者：
Y. Emori;Hiroshi Kawasaki;S. Imajoh;Y. Minami;K. Suzuki
我们已经从 cDNA 序列中确定了钙依赖性蛋白酶内源性抑制剂（CANP 抑制剂，calpastatin）的一级结构，并揭示 CANP 抑制剂包含四个内部重复单元，这可能是其多个反应位点的原因（Emori, Y.、Kawasaki, H.、Imajoh, S.、Imahori, K. 和 Suzuki, K. (1987) Proc. Natl. Acad. Sci. (1987) Proc. Natl. Acad. Sci.美国 84, 3590-3594)。将对应于四个结构域中的每一个的cDNA的限制性片段(编码总共718个残基中的104-156个氨基酸残基)以与载体的lacZ'开放阅读框匹配的方向和框亚克隆到pUC9或pUC18的多克隆位点中。在lac操纵子-启动子系统下，我们成功地在大肠杆菌中产生了CANP抑制剂的截短片段。 CANP抑制剂片段被部分纯化，并检查了对钙依赖性蛋白酶(CANP)的抑制活性。位于四个单元中间的包含约30个氨基酸残基（结构域I-IV）的高度保守区域的所有片段均表现出抑制活性。然而，它们的抑制活性差异很大。进一步的截短实验表明，含有CANP抑制剂30-70个氨基酸残基的小片段仍然保留了抑制活性。从这些实验结果可以得出以下结论：1）CANP抑制剂的四个重复单元（约140个氨基酸残基）中的每一个都是真正的功能单元，可以独立抑制CANP活性； 2) 对应于包含共有 Thr-Ile-Pro-Pro-X-Tyr-Arg 序列的约 30 个氨基酸残基的高度保守序列的结构域对于抑制活性至关重要，并且边界区域对于其调节很重要。
We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.