Ziegler K, Kolac C, Ising W
Institute of Pharmacology and Toxicology, Justus-Liebig-University, Giessen, Germany.
Biochim Biophys Acta. 1994 Dec 30;1196(2):209-17. doi: 10.1016/0005-2736(94)00228-2.
Certain peptide drugs, such as the linear hydrophobic renin-inhibitor EMD 51921, are rapidly eliminated via the bile. At the sinosoidal membrane of liver cells EMD 51921 is taken up via a sodium-independent carrier-mediated mechanism, competing for the uptake of bile acids. Until now, the mechanisms of biliary excretion of EMD 51921 were unknown. In this study we describe an ATP-dependent transport system for the enzymatically and metabolically stable hydrophobic linear renin-inhibiting peptide EMD 51921. The ATP-dependent uptake into the osmotic reactive intravesicular space is saturable (Km 12 microM, Vmax 663 pmol/min per mg protein), temperature dependent and specifically requires ATP. Transport is inhibited by vanadate but not by ouabain, EGTA or NaN3, and does not function in basolateral plasma membrane vesicles. Transport is not altered in canalicular membrane vesicles isolated from Tr- rats lacking the canalicular ATP-dependent transport of cysteinyl leukotrienes and related anions. Transport is inhibited by taurocholate, a typical substrate of the canalicular ATP-dependent bile acid transporter, but also by vincristine and daunomycin, substrates of P-glycoproteins. EMD 51921, however, only inhibits the uptake of taurocholate, whereas the transport of daunomycin is not influenced. Taurocholate and EMD 51921 are mutually non- or un-competitive transport inhibitors. Incubation of rat liver canalicular membranes with micromolar concentrations of EMD 51921 resulted in a 1.8-2.5-fold increase in the rate of ATP-hydrolysis. In contrast, ATP-hydrolysis was not affected by fragments of the peptide that are not transported in an ATP-dependent manner. The apparent Km value (EMD) for ATP-hydrolysis is 68 microM. Vmax is 0.032 U/mg protein. ATPase activity is pH dependent. Stimulation of ATP-hydrolysis is inhibited by vanadate, NEM, hydroxymercuribenzoate and ascorbate, but is not affected by ouabain, EGTA or NaN3. EMD 51921 does not stimulate the ATPase activity of the Na+/K(+)-ATPase isolated from kidney medulla. The EMD-stimulatable ATPase seems to be distinct from the glutathione-S-conjugate stimulatable ATPase and the mdr 1a/b gene products and differs in its characteristics from that of the canalicular ecto-ATPase.
某些肽类药物，如线性疏水肾素抑制剂EMD 51921，可通过胆汁迅速消除。在肝细胞的窦状膜上，EMD 51921通过一种不依赖钠的载体介导机制被摄取，与胆汁酸的摄取相互竞争。到目前为止，EMD 51921的胆汁排泄机制尚不清楚。在本研究中，我们描述了一种针对酶促和代谢稳定的疏水线性肾素抑制肽EMD 51921的ATP依赖性转运系统。ATP依赖性摄取到渗透反应性囊泡内空间是可饱和的（Km为12微摩尔，Vmax为663363皮摩尔/分钟/毫克蛋白质），依赖温度且特别需要ATP。转运受到钒酸盐的抑制，但不受哇巴因、乙二醇双四乙酸或叠氮化钠的抑制，并且在基底外侧质膜囊泡中不起作用。在从缺乏半胱氨酰白三烯和相关阴离子的小管ATP依赖性转运的Tr - 大鼠分离的小管膜囊泡中，转运没有改变。转运受到牛磺胆酸盐（小管ATP依赖性胆汁酸转运体的典型底物）的抑制，但也受到长春新碱和柔红霉素（P - 糖蛋白的底物）的抑制。然而，EMD 51921仅抑制牛磺胆酸盐的摄取，而柔红霉素的转运不受影响。牛磺胆酸盐和EMD 51921是相互非竞争性或非竞争性的转运抑制剂。用微摩尔浓度的EMD 51921孵育大鼠肝脏小管膜导致ATP水解速率增加1.8 - 2.5倍。相反，ATP水解不受不以ATP依赖性方式转运的肽片段的影响。ATP水解的表观Km值（EMD）为68微摩尔。Vmax为0.032 U/毫克蛋白质。ATP酶活性依赖于pH。钒酸盐、N - 乙基马来酰胺、对羟基汞苯甲酸和抗坏血酸抑制ATP水解的刺激，但不受哇巴因、乙二醇双四乙酸或叠氮化钠的影响。EMD 51921不刺激从肾髓质分离的Na+/K(+) - ATP酶的ATP酶活性。EMD刺激的ATP酶似乎与谷胱甘肽 - S - 共轭物刺激的ATP酶和mdr 1a/b基因产物不同，并且其特性与小管外向ATP酶不同。
