Calcium-activated neutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms.
Calcium-activated neutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms.
复制标题
人脑钙激活中性蛋白酶：亚基结构和多种分子形式的酶学特性。
DOI：
10.1111/j.1471-4159.1986.tb00718.x
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发表时间：
1986
影响因子：
4.7
通讯作者：
Nixon,RA
中科院分区：
文献类型：
--
作者：
Vitto,A;Nixon,RA
用二乙氨基乙基(DEAE)-纤维素、苯基琼脂糖凝胶、超凝胶ACA-44和DEAE-Biogel A层析，从人死后大脑皮质中纯化了2,625倍的钙激活中性蛋白酶(CANP)。经Sephacryl 300凝胶过滤，CANP的主要活性形式为94-100kD，由78kD和27kD亚基组成。双向凝胶电泳法将小亚基分解为两个等电点不同的分子物种。CANP能降解大多数人细胞骨架蛋白，但对Fodrin和神经丝蛋白亚基(145kD>；200kD>；70kD)尤其活跃。该酶对[甲基-14C]偶氮酶的半最大激活需要175mMCa~(2+)，对[μ-14C]偶氮酶的最佳活性需要2mM。其他二价金属离子对该酶的激活作用较差，包括铜、铅和锌在内的一些金属离子对该酶有强烈的抑制作用。铝是一种神经毒性离子，在哺乳动物大脑中诱导神经丝堆积，在1mM时抑制47%，在5 mM时抑制100%。在DEAE-Biogel A层析中，缺少27-kD亚基的第二种CANP形式被部分从100-kD异源二聚体中分离出来。78-kD单体与100-kD杂二聚体具有相同的比活力、钙离子需要量、最适pH和对细胞骨架蛋白的专一性，表明27-kD亚基对酶的主要催化性质不是必需的。当CANP接触钙时，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以往报道的不同，后者将其他物种中的mCANP描述为76-80-kD单体或包含76-80-kD和17-20-kD亚基的异源二聚体。100kD人脑CANP与非神经组织CANPs的相似性表明，异二聚体形式在不同组织和物种之间相对保守。
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 > 200 Kd > 70 Kd). The enzyme required 175μMCa2+for half‐maximal activation and 2mM Ca2+for optimal activity toward [methl‐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 mMand 100% at 5mM 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.