Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.
Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.
复制标题
牙龈卟啉单胞菌中两种形式的高分子量半胱氨酸蛋白酶（卟啉蛋白酶）的纯化和表征。
DOI：
10.1128/jb.176.15.4549-4557.1994
复制
发表时间：
1994
影响因子：
3.2
通讯作者：
Lantz,MS
中科院分区：
文献类型：
--
作者：
Ciborowski,P;Nishikata,M;Allen,RD;Lantz,MS
牙龈卟啉单胞菌是一种与人类牙周病的病因和发病机制有关的微生物，它能产生多种强有力的蛋白水解酶，而且这些酶在牙周组织的破坏中起着直接的作用。我们现在报道，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，牙龈假单胞菌W12的两种与细胞相关的半胱氨酸蛋白酶，分子量分别约为150 kDa(Popphypain-1)和120 kDa(Popphypain-2)。这些蛋白酶似乎是一个180 kDa酶的稳定构象变体，它们是迄今为止从牙龈假单胞菌中纯化的最大的半胱氨酸蛋白酶。纯化的蛋白水解酶分别是对甲苯磺酰-甘氨酸-L-Pro-L-精氨酸对硝基苯胺和对硝基苯胺对甲苯磺酰-甘氨酸-L-Pro-L-赖氨酸。合成底物Popphypain-1和-2的水解需要被还原剂激活，被乙二胺四乙酸乙二酯抑制，并在甘氨酸衍生物的存在下被刺激，精氨酸氨解活性对亮蛋白和H-D-酪氨基-L-丙氨基-L-精氨酰氯甲基酮敏感，而赖氨酸氨解活性对对甲苯磺酰-L-赖氨酰氯甲基酮敏感，对亮蛋白不敏感。这些数据表明，孔藻含有两种类型的活性位点。这些细胞相关的牙龈假单胞菌蛋白水解酶可能对牙周组织的破坏有重要和直接的作用。
Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.