Nixon R A, Marotta C A
J Neurochem. 1984 Aug;43(2):507-16. doi: 10.1111/j.1471-4159.1984.tb00928.x.
Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two- to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP break-down products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskeletal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.
组织蛋白酶D（CD）从死后人类大脑皮层中纯化至同质。将CD与人神经丝蛋白（NFPs）一同孵育，这些神经丝蛋白是通过轴突漂浮法制备的，孵育后导致200,000、160,000和70,000道尔顿的神经丝蛋白亚基（200K、160K和70K）迅速降解，这些亚基是通过一维或二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分离得到的。在酶活性与底物蛋白的比例下，降解现象明显，该比例比大脑皮层未分级匀浆中的比例低两到三倍。通过PAGE分离的神经丝蛋白的定量测量表明，在消化早期，160K神经丝蛋白的降解速度比70K亚基稍快，而200K神经丝蛋白的降解速度处于中间水平。在足够高的酶浓度下，人神经丝蛋白制剂中的所有内源性蛋白质都易受组织蛋白酶D的作用。人脑中的组织蛋白酶D也以类似的特异性降解来自小鼠脑的神经丝蛋白制剂中的细胞骨架蛋白。为了鉴定特定的神经丝蛋白降解产物，将针对每种主要神经丝蛋白的抗血清应用于通过二维SDS - PAGE分离的神经丝蛋白的硝酸纤维素电转印膜上。除了检测人神经丝蛋白制剂中的200K、160K和70K神经丝蛋白外，抗血清还检测到了与新鲜大鼠脑的神经丝蛋白制剂中相似的非重叠多肽组。当人神经丝蛋白制剂与组织蛋白酶D孵育时，每个神经丝蛋白亚基会以特定模式释放出额外的多肽。组织蛋白酶D从神经丝蛋白产生的一些免疫交叉反应片段在二维凝胶上与未孵育制剂中存在的多肽共迁移。这些结果表明神经丝蛋白和其他细胞骨架蛋白是组织蛋白酶D的底物。本文讨论了这些发现的生理学意义以及分析蛋白质降解产物对于确定体内蛋白酶作用的可能用途。
