Nixon R A, Quackenbush R, Vitto A
J Neurosci. 1986 May;6(5):1252-63. doi: 10.1523/JNEUROSCI.06-05-01252.1986.
Calcium-activated neutral proteinases (CANPs) and their specificities for axonally transported proteins were studied within intact axons of mouse retinal ganglion cell (RGC) neurons in vitro. Two CANP activities with markedly different properties were identified. CANP B, at endogenous calcium levels, selectively cleaved the 145,000 Da (145 kDa) neurofilament protein subunit to yield 143 and 140 kDa neurofilament proteins that are also major constituents of the axonal cytoskeleton. This process represents a posttranslational modification of the neurofilament protein subunit rather than the initial step in its degradation (Nixon et al., 1982, 1983). A second calcium-activated neutral proteinase activity, CANP A, appeared only when calcium levels in the incubating medium were 100 microM or higher. CANP A degraded most proteins in RGC axons but acted considerably more rapidly on high-molecular-weight species. In particular, a 290-320 kDa protein in the Group IV (SCb) phase of axoplasmic transport was degraded 3 X faster than other major axonal proteins, including neurofilament proteins and fodrin. When maximally expressed, CANP A activity represented an enormous proteolytic potential in RGC axons--more than 50% of the total axonal content of proteins larger than 60 kDa could be hydrolyzed within 5 min. The calcium requirements, inhibitor profile, and substrate specificity of CANP A were similar to those of mCANP, the major CANP of mouse brain purified to homogeneity, suggesting that these enzymes may be the same or highly related proteins. The existence in a single neuron type of two CANP activities with markedly different substrate specificities and enzymatic properties emphasizes the possible functional diversity of calcium-activated neutral proteinases in neurons. These functions include the posttranslational modification, as well as degradation of neuronal proteins.
在体外对小鼠视网膜神经节细胞（RGC）神经元的完整轴突内的钙激活中性蛋白酶（CANPs）及其对轴突运输蛋白的特异性进行了研究。鉴定出了两种性质明显不同的CANP活性。在生理钙水平下，CANP B选择性地切割145,000 Da（145 kDa）神经丝蛋白亚基，产生143 kDa和140 kDa的神经丝蛋白，它们也是轴突细胞骨架的主要成分。这个过程代表了神经丝蛋白亚基的翻译后修饰，而不是其降解的起始步骤（尼克松等人，1982年，1983年）。第二种钙激活中性蛋白酶活性，CANP A，仅在孵育培养基中的钙水平为100 microM或更高时出现。CANP A降解RGC轴突中的大多数蛋白质，但对高分子量物质的作用要快得多。特别是，轴浆运输IV组（SCb）阶段的一种290 - 320 kDa的蛋白质比其他主要轴突蛋白，包括神经丝蛋白和血影蛋白，降解速度快3倍。当最大程度表达时，CANP A活性在RGC轴突中代表了巨大的蛋白水解潜力——在5分钟内，大于60 kDa的蛋白质的总轴突含量的50%以上可以被水解。CANP A的钙需求、抑制剂谱和底物特异性与mCANP相似，mCANP是从小鼠脑中纯化至同质的主要CANP，这表明这些酶可能是相同或高度相关的蛋白质。在单一神经元类型中存在两种底物特异性和酶学性质明显不同的CANP活性，强调了钙激活中性蛋白酶在神经元中可能的功能多样性。这些功能包括翻译后修饰以及神经元蛋白质的降解。
