Lalitha K, Easwari K
Department of Chemistry, Indian Institute of Technology, Madras, India.
Biol Trace Elem Res. 1995 Apr;48(1):67-89. doi: 10.1007/BF02789080.
Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.
我们实验室早期的研究表明，硒对缺硒豆类作物绿豆在发芽过程中具有有益作用，这在与生长相关的参数以及75硒的特定吸收中得到体现。从在对照（无硒）和补充硒（0.5、1.0和2.0 ppm硒）条件下发芽的幼苗中分离出的线粒体对体外添加的Na2(75)SeO3的吸收表明，在浓度高达25 microM时，随着Na2(75)SeO3添加量的增加，吸收量呈比例增加。75硒的吸收在长达15分钟的时间内随时间呈线性增加，在30分钟时出现明显的外流。结果表明在硒运输过程中存在协同效应。在整个线粒体和线粒体蛋白组分中，对15分钟和30分钟时间间隔内75硒的吸收进行了动力学分析。使用Lineweaver-Burk图、Hill图、log [v]对log [A]以及Scatchard图进行的图形分析证实，在75硒吸收过程中存在负协同效应。Hill系数（nH）值估计约为0.7 - 0.8。75硒吸收的Scatchard图是双相的，表明可能存在两类结合位点。高亲和力和低亲和力结合位点的数量估计分别约为4 - 7和26 - 30 nmol/mg蛋白。使用线粒体呼吸抑制剂的研究表明，总75硒吸收量的约10 - 20%是能量依赖性的。硫酸盐和亚硫酸盐（5 - 25 microM）对75硒吸收的抑制约60 - 70%，这意味着二羧酸转运体参与了硒的运输。CdCl2、HgCl2、汞撒利和NEM使75硒吸收减少40 - 60%，证实了巯基在该过程中的相互作用。本研究得出了绿豆线粒体对75硒吸收具有调节性质的证据。
