Parenti P, Villa M, Hanozet G M
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
J Biol Chem. 1992 Aug 5;267(22):15391-7.
The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.
利用刷状缘膜囊泡研究了鳞翅目幼虫中肠K⁺-亮氨酸共转运的动力学。在类似于体内发生的实验条件下，即在膜电位差（Δψ）远大于0（内侧为负）且pH梯度为1.4个单位（胞内pH 7.4/胞外pH 8.8）的情况下，测定亮氨酸摄取的初始速率（3秒）。亮氨酸和K⁺根据顺序机制与载体结合，一种底物的结合使另一种底物的解离常数改变了0.15倍。反式K⁺和反式亮氨酸都是亮氨酸摄取的混合型抑制剂。此外，总亮氨酸摄取的一部分不依赖K⁺，且被反式亮氨酸竞争性抑制。我们将反式抑制作用解释为意味着仅部分装载K⁺的形式实际上无法跨膜转运，而二元复合物载体亮氨酸可以从膜的反式侧异构化为顺式侧。然而，不依赖K⁺的亮氨酸摄取发生时的平衡常数大于1，即通过仅部分装载亮氨酸的形式的外流途径比空载载体从反式侧到顺式侧的异构化速率慢。综上所述，这些结果提示了一种通过等随机双底物双位点系统进行转运的模型。由于K⁺并非作为纯粹的竞争性激活剂起作用，该模型不同于为大多数与Na⁺相关的溶质转运机构所提出的模型，并且可能与鳞翅目幼虫中氨基酸转运体阳离子特异性的拓宽有关。
