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

(1)我们进行了模型实验，其中部分纯化的氨基酰tRNA合成酶与活性氧产生系统孵育。一部分酶变得热不稳定，因此在热失活方面模仿“旧”酶。因此，我们认为活性氧可能参与了改变蛋白的产生。(2)我们证明了在原代培养中微注射到小鼠肝细胞中的蛋白质以及细胞内标记的蛋白质在老年动物细胞中的半衰期延长了50%至60%。因此，我们认为，衰老动物体内改变蛋白的积累部分是由于蛋白质周转的减少。(3) 23月龄小鼠限食2个月，测定热不稳定酶的比例和蛋白质半衰期。热不稳定酶的比例降低到幼龄动物的水平，蛋白质的半衰期也降低了。因此，这表明饮食限制的抗衰老作用至少部分是“恢复”随着年龄增长而减少的蛋白质代谢。(4)利用抗泛素抗体测定了幼龄和老年小鼠脑、肝、肾中游离泛素和结合泛素的含量。老年动物大脑中游离泛素的数量减少了约40%，但其他组织中没有。泛素的共轭形式在年老的大脑中显著增加，但在其他组织中没有。因此，在年老动物的大脑中，被改变的蛋白质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.