Vitto A, Nixon R A
J Neurochem. 1986 Oct;47(4):1039-51. doi: 10.1111/j.1471-4159.1986.tb00718.x.
Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94-100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd greater than 200 Kd greater than 70 Kd). The enzyme required 175 microM Ca2+ for half-maximal activation and 2 mM Ca2+ for optimal activity toward [methyl-14C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM. A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.
通过在二乙氨基乙基（DEAE）-纤维素、苯基琼脂糖、Ultrogel AcA - 44和DEAE - 生物凝胶A上进行层析的方法，从人死后的大脑皮层中纯化出钙激活中性蛋白酶（CANP），纯化倍数为2625倍。通过在Sephacryl 300上进行凝胶过滤，CANP的主要活性形式显示分子量为94 - 100千道尔顿（Kd），由78 - Kd和27 - Kd亚基组成。二维凝胶电泳将小亚基解析为两种具有不同等电点的分子种类。CANP能降解大多数人细胞骨架蛋白，但对血影蛋白和神经丝蛋白亚基（145 Kd＞200 Kd＞70 Kd）尤其具有活性。该酶激活的半最大活性需要175微摩尔/升的Ca²⁺，对[甲基 - ¹⁴C]偶氮酪蛋白具有最佳活性则需要2毫摩尔/升的Ca²⁺。其他二价金属离子是该酶的不良激活剂，其中一些，包括铜、铅和锌，能强烈抑制该酶。铝是一种在哺乳动物脑中诱导神经丝积聚的神经毒性离子，在1毫摩尔时抑制该酶47%，在5毫摩尔时抑制100%。在DEAE - 生物凝胶A层析过程中，从100 - Kd异二聚体中部分分离出了一种缺少27 - Kd亚基的第二种CANP形式。78 - Kd单体与100 - Kd异二聚体表现出相同的比活性、钙离子需求、最适pH以及对细胞骨架蛋白的特异性，这表明27 - Kd亚基对于该酶的主要催化特性并非必不可少。当CANP暴露于钙时，27 - Kd亚基迅速自溶为18 - Kd中间体，这可能解释了我们的结果与先前报告之间的差异，先前报告将其他物种中的脑mCANP描述为76 - 80 - Kd单体或包含76 - 80 - Kd和17 - 20 - Kd亚基的异二聚体。100 - Kd人脑中的CANP与非神经组织中的CANP的相似性表明，异二聚体形式在各种组织和物种中相对保守。
