Zoccoli M A, Hoopes R R, Karnovsky M L
J Biol Chem. 1982 Oct 10;257(19):11296-300.
Intact microsomes from groups of fed, fasted, glucocorticoid-treated (triamcinolone) and diabetic (alloxan) rats were reacted with 4,4'-diisothiocyanostilbene-2,2'-disulfonic (DIDS), a specific inhibitor of microsomal glucose-6-P translocase. The concentrations that inhibit by 50% were 41 +/- 2, 31 +/- 1, 39 +/- 4, and 18 +/- 1 microM (mean +/- S.E.; n = 3); (order as above). The maximal levels of inhibition of the translocase by DIDS were 66 +/- 2, 79 +/- 2, 63 +/- 1, and 88 +/- 1%, respectively. The differences in the values for the different groups of animals are statistically significant, except for comparisons between fed and triamcinolone-treated animals. Microsomes from the same groups of animals were treated with the tritiated reduced derivative of DIDS, [3H]H2DIDS, which labels a 54,000-dalton polypeptide, previously implicated as a component of the glucose-6-P translocase. The mean values (+/- S.E.) of [3H]H2DIDS bound to the polypeptide under saturating conditions were 100 +/- 6, 120 +/- 9, 62 +/- 7, and 101 +/- 15 pmol/mg of microsomal protein, respectively. The amount bound in microsomes from triamcinolone-treated rats is significantly lower from the values for the other three physiological states, which do not differ significantly from each other. The presence of glucose-6-P, but not mannose-6-P, during the [3H]H2DIDS reaction significantly stimulates the labeling of the 54,000-dalton polypeptide in microsomes from all the classes of animals above, except the diabetic animals. These results indicate that DIDS and [3H]H2DIDS are probes sensitive enough to discern differences in the translocase due to physiological regulation. On the basis of the labeling studies with [3H]H2DIDS, the increase in translocase activity observed in microsomes from fasted, triamcinolone-treated, and diabetic rats cannot be ascribed to increased numbers of translocase molecules, but rather to increased functional activity of the translocase protein.
将喂食、禁食、糖皮质激素处理（曲安西龙）和糖尿病（四氧嘧啶）大鼠的完整微粒体与微粒体葡萄糖-6-磷酸转运体的特异性抑制剂4,4'-二异硫氰酸芪-2,2'-二磺酸（DIDS）反应。抑制50%的浓度分别为41±2、31±1、39±4和18±1微摩尔（平均值±标准误；n = 3）；（顺序如上）。DIDS对转运体的最大抑制水平分别为66±2、79±2、63±1和88±1%。不同组动物的值差异具有统计学意义，但喂食组和曲安西龙处理组动物之间的比较除外。用DIDS的氚标记还原衍生物[3H]H2DIDS处理来自相同组动物的微粒体，该衍生物标记一种54000道尔顿的多肽，先前被认为是葡萄糖-6-磷酸转运体的一个组成部分。在饱和条件下与该多肽结合的[3H]H2DIDS的平均值（±标准误）分别为100±6、120±9、62±7和101±15皮摩尔/毫克微粒体蛋白。曲安西龙处理大鼠的微粒体中结合的量明显低于其他三种生理状态的值，而其他三种生理状态之间没有显著差异。在[3H]H2DIDS反应过程中，葡萄糖-6-磷酸（而非甘露糖-6-磷酸）的存在显著刺激上述所有类动物（糖尿病动物除外）微粒体中54000道尔顿多肽的标记。这些结果表明，DIDS和[3H]H2DIDS是足够敏感的探针，能够辨别由于生理调节导致的转运体差异。基于用[3H]H2DIDS进行的标记研究，在禁食、曲安西龙处理和糖尿病大鼠的微粒体中观察到转运体活性增加，这不能归因于转运体分子数量的增加，而应归因于转运体蛋白功能活性的增加。
