Svardal A, Refsum H, Ueland P M
J Biol Chem. 1986 Mar 5;261(7):3156-63.
Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.
最近在小鼠和大鼠的各种组织的酸提取物中发现了低浓度（0.5 - 6 nmol/g）的同型半胱氨酸（Hcy）（厄兰，P.M.，赫兰德，S.，布罗克，O.-J.，和尚切，J.-S.（1984年）《生物化学杂志》259，2360 - 2364）。这被称为组织中的游离Hcy。本文描述了一种测定与蛋白质结合的Hcy的方法，该方法包括用硫酸铵沉淀和洗涤组织蛋白，在二硫苏糖醇存在下从天然蛋白质中释放Hcy，以及通过灵敏的放射酶法测定游离Hcy。在动物死亡后的几秒钟内，大鼠组织中的游离和结合Hcy均显著下降。与蛋白质结合的Hcy量在肝脏中最高，在肾脏、大脑、心脏、肺和脾脏中略低。除小脑外，大多数组织中游离Hcy与结合Hcy的比例在1到2之间，小脑中游离Hcy大量过剩（游离/结合比例为18）。游离Hcy几乎完全定位于大鼠肝脏的可溶部分，而与蛋白质结合的Hcy在该部分和微粒体之间的分布大致相等。分离的大鼠肝细胞中游离和与蛋白质结合的Hcy比例与全肝中观察到的比例相同，但大量的Hcy被分泌到细胞外介质中。用[35S]甲硫氨酸进行脉冲追踪实验测定的半衰期，S - 腺苷甲硫氨酸为53秒，S - 腺苷同型半胱氨酸为2秒，Hcy（游离和结合视为一个单一库）为3秒。此外，在整个快速追踪期内这些代谢物之间以及游离和结合Hcy之间的同位素平衡表明，S - 腺苷同型半胱氨酸和Hcy的周转率受生成速率限制，并且Hcy - 蛋白质复合物的解离速率可能大大超过Hcy的周转率。因此，Hcy的半衰期使得游离和结合Hcy参与代谢调节都是可行的。
