Mentlein R, Struckhoff G
Anatomisches Institut, Universität Kiel, F.R.G.
J Neurochem. 1989 Apr;52(4):1284-93. doi: 10.1111/j.1471-4159.1989.tb01877.x.
From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.
从大鼠脑匀浆的可溶性部分和膜部分中，纯化出两种能从生色底物中释放Xaa-Pro型二肽的酶，使其达到均一状态。这两种分离得到的二肽基肽酶具有相似的分子和催化特性：对于天然蛋白质，估计分子量为110,000；对于变性蛋白质，估计分子量为52,500。可溶性肽酶在分析等电聚焦后产生一条pI为4.2的条带，而对于膜相关形式，在pI 4.2和4.3之间检测到另外两条酶活性条带。根据神经氨酸酶处理后相同的图谱判断，两种肽酶均不含唾液酸。估计水解甘氨酰-脯氨酸-和精氨酰-脯氨酸-硝基苯胺的最适pH为5.5。在倒数第二个位置用丙氨酸代替脯氨酸的底物以相当的速率被水解。与具有未封闭的中性或特别是碱性末端的相应底物相比，底物最终N末端位置的酸性氨基酸使肽酶的活性降低100倍。研究了二肽基肽酶对几种具有N末端Xaa-Pro序列的肽的作用。三肽被迅速水解，但随着肽链长度的增加，活性显著降低。尽管四肽P物质1-4仍然是一种良好的底物，但从P物质本身或酪蛋白吗啡中连续释放Xaa-Pro二肽所检测到的活性要低得多。更长的肽不被切割。肽酶能水解Pro-Pro键，例如在缓激肽1-3或1-5片段中，但缓激肽本身具有抗性。这些酶被丝氨酸蛋白酶抑制剂（如二异丙基氟磷酸或苯甲基磺酰氟）和高盐浓度抑制，但不被氨肽酶抑制剂杆菌肽和贝他汀抑制。基于分子和催化特性，这两种酶可归类为二肽基肽酶II（EC 3.4.14.2）而不是IV（EC 3.4.14.5）的种类。然而，一些催化特性使脑内的这种酶与其他来源的二肽基肽酶II形式有所不同。
