Experimental device designed for assay and purification of oxygen-sensitive enzymes and its application.

制造了一个装有净化装置和分光光度计的厌氧手套箱作为试验并投入使用。下面是几个有趣的发现。丙酮酸甲酸裂解酶（PFL）在口腔链球菌的无氧糖代谢中起重要作用。PFL对氧极其敏感，并被认为在催化位点附近含有一个蛋白质自由基。分子氧与自由基的反应似乎进一步产生有害的活性氧，并迅速导致酶的不可逆失活。酶的可逆失活形式是氧相关的，因为它不含自由基，可以被pfl激活酶激活。部分纯化的PFL活化酶不稳定，但与铁离子、硒离子、蝶呤孵育后可恢复部分活性。这种酶似乎含有“一种类似蝶呤的辅因子”，这种辅因子被认为存在于黄嘌呤氧化酶中。变形链球菌的激活酶是由丙酮酸触发的，而不是由草酸触发的，而且很不稳定。链球菌酶的性质被认为与大肠杆菌不同。在厌氧条件下，斑块微生物保持细胞内NADH/NAD平衡是必不可少的。口腔链球菌采用“PFL系统”保持平衡。乳酸杆菌将乙酸代谢成乙醇，氧化NADH保存。在碳酸氢盐存在的情况下，放线菌可以通过琥珀酸途径再生NAD，提高产酸速率。

An anaerobic glove box, equipped with a purification apparatus and a spectorophotometer, was manufactured as a trial and employed. Several interesting finding were turned out as follows.Pyruvate formate-lyase (PFL) plays an important role in anaerobic sugar metabolism in oral streptococci. PFL was extremely sensitive to oxygen, and assumed to contain a protein radical near a catalytic site. The reaction of molecular oxygen with the radical seemed to generate further harmful active oxygen and bring about irreversible inactivation of the enzyme rapidly. Reversible inactive form of the enzyme was oxygen torelant since it did not contain the radical, and could be activated by PFL-activating enzyme. Partially purified PFL activating enzyme was quite unstable, but incubation of the enzyme with ferrous ion, selenium ion, pterin, restored a part of the activity. The enzyme seemed to contain "a pterin like cofactor" which was assumed to be exist in xanthine oxidase. The activating enzyme of Streptococcus mutans was triggered by pyruvate, but not oxamate, and quite unstable. The properties of the streptococcal enzyme is considered different from that of Escherichia coli.It is essential for plaque microorganisms to keep intracellular NADH/NAD balance under anaerobic conditions. Oral streptococci employed "the PFL system" to keep the balance. Lactobacilli metabolized acetate into ethanol to oxidize NADH to keep it. Actinomyces could regenerate NAD via succinate pathway when bicarbonate was available, and increased the rate of acid production.