Cotgreave I A, Weis M, Berggren M, Sandy M S, Moldéus P W
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
J Biochem Biophys Methods. 1988 Aug;16(4):247-54. doi: 10.1016/0165-022x(88)90058-9.
The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.
用单溴代双硫腙对完整的大鼠肝细胞进行衍生化处理，可使几个亚细胞组分中可及的蛋白质巯基快速标记。衍生化过程不会引起急性细胞毒性，也不会改变各组分的浮力密度或其总蛋白质组成。对与各组分不可逆结合的荧光进行定量分析，结果表明该巯基库在细胞内存在显著的异质性。巯基含量在胞质溶胶中最高（约90 nmol/mg蛋白质），在微粒体（约65 nmol/mg蛋白质）和线粒体（约45 nmol/mg蛋白质）中居中，而在含有细胞核和质膜的粗组分中最低（约35 nmol/mg蛋白质）。对分级分离后衍生化的微粒体和胞质溶胶得到了相似的值，但分离的线粒体和核/质膜组分的相应值显著增加了约100%。本文从分级分离过程中单溴代双硫腙蛋白质巯基的动态通量以及这种非侵入性方法在研究反应性外来化合物的毒性机制和蛋白质巯基在正常细胞功能中的作用方面的适用性进行了讨论。
