Investigation of proteases responsible for degradation of periodontal tissue by periodontopathogens

1.中间普雷沃氏菌弹性分解酶从成人牙周病变脓液中分离到中间普雷沃氏菌弹性分解菌株。弹性酶与包膜相结合，可与胍-盐酸溶解。通过离子交换层析，凝胶过滤和疏水相互作用层析等顺序程序纯化酶至均匀性。该弹性蛋白酶为丝氨酸蛋白酶，质量为31 kDa。该酶能水解弹性蛋白粉，但不能降解胶原蛋白和偶氮偶联蛋白。两种合成的胰腺底物（戊二酰- ala - ala -亲亮氨酸对硝基苯胺）和白细胞弹性酶（甲氧基琥珀酰- ala - ala -亲亮氨酸对硝基苯胺）均被水解。从牙周病患者的牙菌斑样本中，我们分离出对琥珀酸- ala - ala - pro- phe -硝基苯胺（乳糜蛋白酶底物）和明胶具有蛋白水解活性的厌氧菌株。然而，只有当有机体在补充全血的培养基中培养时，才观察到这种蛋白质水解。这类菌株的蛋白酶可能受到血液成分的刺激或诱导。这些菌株是厌氧革兰氏阳性棒，在一半的检查斑块样本中发现。它们的共同特性是：对过氧化氢酶和吲哚的生产、硝酸盐还原、胰岛素水解、葡萄糖和乳糖的发酵均有抑制作用。乙酸被检测为代谢产物。这些发现表明分离物类似于非破糖真细菌。血液对蛋白酶产生的影响可替代血清、血清白蛋白和唾液。纯化后的蛋白酶可水解戊二酸- ala - ala -前亮氨酸对硝基苯胺和偶氮唑，以及上述的胰凝乳蛋白酶底物和明胶。其质量为150 kDa，活性被苯基甲基磺酰氟抑制，表明该酶为丝氨酸蛋白酶。

1.Elastolytic enzyme of prevotella intermediaElastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-Ala-Ala-Pro-Leu P-nitroanilide) and leukocyte elastase (methoxy succinyl-Ala-Ala-Pro-Val p-nitroanilide) were hydrolyzed.Protease of Eubacterium like organismFrom the dental plaque samples of periodontal patients, we isolated anaerobic strains displaying proteolytic activity against succiny-Ala-Ala-Pro-Phe P-nitroanilide (chymotrypsin substrate) and gelatin. However, this proteolysis was observed only when the organisms were cultured in the media supplemented with whole blood. The protease of such strains may be stimulated or induced by component(s)of blood. These strains were anaerobic gram-positive rods and were found in half the examined plaque samples. Their common properties were as follows : nogative in production of catalase and indole, nitrate reduction, esculin hydrolysis, and fermentation of glucose and lactose. Acetic acid was detected as a metabolic product. These findings suggest that the isolates resemble nonsaccharoclastic Eubacterium. The effect of blood on protease production could be substituted for serum, serum albumin, and saliva. The purified protease hydrolyzed glutary-Ala-Ala-Pro-Leu p-nitroanilide and azocoll as well as the chymotrypsin substrate described above and gelatin. Its mass was 150 kDa and the activity was inhibited by phenylmethylsulfonyl-fluoride, indicating this enzyme is a serine protease.