Studies on the effect of oxygensensitive enzymes on ecology and acid production of dental plaque.

三磷酸核糖核苷还原酶是厌氧微生物中唯一催化合成脱氧核糖核苷酸反应的酶。这种酶是这些微生物生长和生存所必需的酶，因为脱氧核糖核苷酸是DNA合成的材料。这种酶的活性已经用放射性同位素测量过了。然而，准确测量活性是困难的，因为这种酶对氧极其敏感，在厌氧手套箱中使用放射性同位素在法律上是困难的，特别是在日本。我们首先可以建立一种以二苯胺为显色剂的光度法测定该酶活性的方法。用这种方法，我们发现这种酶在口腔微生物中的活性，变形链球菌，一种强致龋菌。为了更准确地估计活性，我们使用了高效液相色谱法。然后，我们发现s.t amans中的酶也是高度氧敏感的，并且这种酶具有可逆的失活形式。这种酶的失活形式被另一种对氧高度敏感的酶——丙酮酸甲酸裂解酶——以类似的方式激活。可能是同一种激活酶激活了这两种氧敏感酶。这些发现表明，牙菌斑中可变的氧浓度强烈地影响这些酶的活性，然后影响与龋齿和牙周病的发生和发展有关的微生物。

Ribonucleoside triphosphate reductase is an only one enzyme that catalyzes the reaction to synthesize deoxyribonucleotides in anaerobic microorganisms. This enzyme is an essential enzyme for growth and survival of these microorganisms, because deoxyribonucleotides are material for DNA synthesis. The activity of this enzyme has been measured with radio isotopes. However, the accurate measurement of the activity was diffcult, because this enzyme is extremely oxygen-sensitive and usage of radioisotopes in an anaerobic glove box is legally difficult especially in Japan. We could first develop a method to estimate the activity of this enzyme by photometric method with diphenylamine as a color producing reagent in the cell-free extracts of Escherichia coli. With this method we then find the activity of this enzyme in an oral microorganism, Streptococcus mutans, a strong cariogenic bacterium. To estimate the activity more accurately, we uesd high performance liquid chromatography. Then, we found that the enzyme in S.mutans is also highly oxygen-sensitive and that this enzyme has a reversible inactive form. The inactive form of this enzyme was activated by a similar way as found in another highly oxygen-sensitive enzyme, pyruvate formate-lyase. It is possible that the same activating enzyme activates these two oxygen-sensitive enzymes. These findings sugggest that the variable oxygen concentration in dental plaque affects strongly the activities of these enzymes and then the microorganisms involved in the initiation and development of dental caries as well as periodontal diseases.