Vincenzini M T, Favilli F, Iantomasi T
Institute of Biochemistry, University of Florence, Italy.
Biochim Biophys Acta. 1988 Jul 7;942(1):107-14. doi: 10.1016/0005-2736(88)90279-9.
Glutathione transport was studied in brush-border membrane vesicles of rabbit small intestine in which gamma-glutamyl transpeptidase (EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT125). Transport of intact [glycine-2-3H]GSH occurred into an osmotically active intravesicular space of AT125-treated membranes. The 0.1 M NaSCN gradient (Na+ inside greater than Na+ outside) in the transport medium could be replaced with KSCN or NaCl without affecting transport activity. The initial rate of GSH transport followed Michaelis-Menten saturation kinetics (Km = 17 microM). The results suggest that, in these membranes, there was an Na+-independent mediated transport for intact GSH with marked specificity and affinity. In fact glycine, glutamic acid and cysteine did not decrease GSH uptake, as was also true for glycylglycine and glycylglycylglycine; only gamma-glutamylcysteinylglycyl ester, a derivative of GSH, partially inhibited GSH transport.
利用特异性亲和标记试剂L-(αS,5S)-α-氨基-3-氯-4,5-二氢-5-异恶唑乙酸(AT125)使兔小肠刷状缘膜囊泡中的γ-谷氨酰转肽酶(EC 2.3.2.2)失活，在此基础上研究了谷胱甘肽转运。完整的[甘氨酸-2-³H]谷胱甘肽可转运至经AT125处理的膜囊泡具有渗透活性的囊泡内空间。转运介质中的0.1 M硫氰酸钠梯度(囊泡内Na⁺浓度高于囊泡外)可用硫氰酸钾或氯化钠替代，而不影响转运活性。谷胱甘肽转运的初始速率符合米氏饱和动力学(Km = 17 μM)。结果表明，在这些膜中，存在一种不依赖Na⁺的介导转运，可转运完整的谷胱甘肽，且具有显著的特异性和亲和力。实际上，甘氨酸、谷氨酸和半胱氨酸不会降低谷胱甘肽的摄取，甘氨酰甘氨酸和甘氨酰甘氨酰甘氨酸也是如此；只有谷胱甘肽的衍生物γ-谷氨酰半胱氨酰甘氨酸甲酯可部分抑制谷胱甘肽转运。
