Biochemical studies on the mechanism of Generation and accumulation of altered enzymes with age

(1)我们进行了部分纯化的氨基酰tRNA合成酶与活性氧产生系统孵育的模型实验。酶的一部分变得不耐热，因此在热灭活谱方面模仿了“旧的”酶。结果表明，原代培养小鼠肝细胞显微注射蛋白和细胞内标记蛋白的半衰期在老年动物肝细胞中延长了50%~60%。23月龄小鼠限食2个月后，测定不耐热酶的比例和蛋白质的半衰期。热不稳定酶的比例降低到幼年动物的水平，蛋白质的半衰期也是如此。因此，我们认为限制饮食的抗衰老作用至少部分是为了使随着年龄增长而出现的蛋白质代谢下降的蛋白质代谢恢复活力。(4)利用抗泛素抗体，我们测定了幼年和老年小鼠脑、肝脏和肾脏中游离泛素和结合泛素的含量。老年动物大脑中的游离泛素含量减少了约40%，但其他组织中的含量却没有下降。结合形式的泛素在老年人的大脑中显著增加，但在其他组织中则没有。因此，改变的蛋白质在老年动物的大脑中的周转被认为是减少的。

(1) We performed model experiments where partially purified aminoacyl tRNA synthetases are incubated with active oxygen producing systems. A part of the enzymes was rendered heat-labile, thus mimicking "old" enzymes in terms of heat-inactivation profiles. It is therefore suggested that active oxygens might be involved in the generation of altered proteins.(2) We demonstrated that half-lives of proteins microinjected into mouse hepatocytes in primary culture as well as intracellularly labeled proteins are extended by 50 to 60% in the cells from old animals. It is therefore suggested that accumulation of altered proteins in senescent animals is partly due to decrease in the turnover of proteins.(3) Twenty third month-old mice were subjected to dietary restriction for 2 months, and then proportion of heat-labile enzymes and half-life of proteins were examined. Proportion of heat-labile enzymes was decreased to a level of young animals and so are the half-lives of proteins.Thus it is suggested that anti-aging effect of dietary restriction is at least partly to "rejuvenate" decreased protein metabolism that occur with age.(4) Using anti-ubiquitin antibody we measured amounts of free and conjugated ubiquitin in the brain, liver and kidney of young and old mice. The amount of free ubiquitin in the brain was decreased by about 40% in the old animals but not in the other tissues. Conjugated forms of ubiquitin was increased significantly in the old brains but not in other tissues. The turnover of altered proteins is thus suggested to decrease in the brain of old animals.