Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain.
Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain.
复制标题
钙激活中性蛋白酶 (mCANP) 的免疫测定和活性：人和小鼠脑中可溶性和膜相关部分的分布。
DOI：
10.1111/j.1471-4159.1992.tb11374.x
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发表时间：
1992
影响因子：
4.7
通讯作者：
Nixon,RA
中科院分区：
文献类型：
--
作者：
Takeuchi,KH;Saito,KI;Nixon,RA
毫米级钙激活中性蛋白酶(MCANP)通常被认为是非神经系统中的一种胞浆酶，尽管它在大脑中的亚细胞定位还不是很清楚。为了解决关于基于活性测量的mCANP定位的相互矛盾的报道，我们建立了CANP的免疫分析方法，并比较了小鼠和人脑的可溶性和膜部分中该分子的含量和活性。用两种不同的mCANP抗体进行免疫印迹分析，结果表明mCANP在人脑和小鼠脑中的含量为4.5ngg，约占总蛋白的0.0005%。经15000g离心后，总免疫活性mCANP的95%以上仍留在可溶性部分。以[14C]偶氮酶为底物，去除内源CANP抑制剂S，用DEAE-纤维素柱交换层析测定mCANP活性。仅在从上清液中提取的组分中检测到酪蛋白溶解活性。用不同浓度的Triton X-100提取组织，mCANP含量和酶活性的分布没有变化。这些发现证实了CANP免疫分析的实用性和有效性，并证明了mCANP在小鼠和人脑中主要定位于胞浆中。
The millimolar form of calcium‐activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000gcentrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion‐exchange chromatography on DEAE‐cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X‐100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.