Investigation of proteases responsible for degradation of periodontal tissue by periodontopathogens

1.中间普氏杆菌弹性溶酶从成人牙周病变脓液中分离得到中间普氏杆菌弹性溶酶菌株。弹性蛋白酶与包膜结合，并能被盐酸胍溶解。通过离子交换层析、凝胶过滤和疏水相互作用层析等步骤对该酶进行了纯化。该弹性酶为丝氨酸蛋白酶，分子量为31 kDa。该酶能降解弹性蛋白粉，但不能降解胶原蛋白和偶氮染料结合蛋白。人胰腺合成底物(戊二醛-丙氨酸-丙氨酸-对硝基苯胺)和白细胞弹性酶(甲氧基-琥珀酰丙氨酸-对硝基苯胺)均被水解。然而，只有在添加了全血的培养基中才能观察到这种蛋白质分解。这些菌株的蛋白水解酶可被血液成分(S)刺激或诱导。这些菌株是厌氧革兰氏阳性杆菌，在检查的斑块样本中有一半被发现。它们的共同性质是：能产生过氧化氢酶和吲哚，硝酸盐还原，七叶皂苷的水解，葡萄糖和乳糖的发酵。检测到醋酸是一种代谢产物。这些发现表明，这些分离物类似于非破糖真细菌。血液对蛋白水解酶产生的影响可以替代血清、血清白蛋白和唾液。纯化的蛋白水解酶能降解谷氨酸-丙氨酸-丙氨酸-Pro-亮氨酸、对硝基苯胺和偶氮苯酚、上述胰凝乳酶底物和明胶。该酶的分子量为150 kDa，其活性被苯甲基磺酰氟抑制，表明该酶是一种丝氨酸蛋白酶。

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.