Investigation of proteases responsible for degradation of periodontal tissue by periodontopathogens

1.中间普氏菌的弹性溶解酶从成人牙周病变的脓液样品中分离中间普氏菌的弹性溶解菌株。发现弹性蛋白酶与包膜结合，并且它可以被盐酸胍增溶。通过离子交换层析、凝胶过滤和疏水相互作用层析将酶纯化至均一。该弹性蛋白酶是丝氨酸蛋白酶，其质量为31 kDa。它水解弹性蛋白粉，但胶原蛋白和偶氮共轭蛋白不被这种酶降解。从牙周病患者的牙菌斑中分离出对琥珀酰-Ala-Ala-Pro-Phe对硝基苯胺（糜蛋白酶底物）和明胶具有蛋白水解活性的厌氧菌株，并与白细胞弹性蛋白酶（甲氧基琥珀酰-Ala-Ala-Pro-瓦尔对硝基苯胺）进行比较。然而，这种蛋白水解仅在生物体在补充有全血的培养基中培养时观察到。这些菌株的蛋白酶可以由血液的组分刺激或诱导。这些菌株是厌氧革兰氏阳性杆菌，在一半的检查菌斑样本中发现。它们的共同特性是：过氧化氢酶和吲哚的产生、硝酸盐还原、七叶苷水解、葡萄糖和乳糖发酵均为阴性。乙酸被检测为代谢产物。这些发现表明分离物类似于非糖杆菌真杆菌。血液对蛋白酶产生的影响可以替代血清、血清白蛋白和唾液。纯化的蛋白酶水解戊二酰-Ala-Ala-Pro-Leu对硝基苯胺和azocoll以及上述胰凝乳蛋白酶底物和明胶。该酶分子量为150 kDa，其活性可被苯甲磺酰氟抑制，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.