Department of Biology, 1 UNF Drive, University of North Florida, Jacksonville, FL 32224, USA.
J Exp Biol. 2010 Mar 1;213(5):769-74. doi: 10.1242/jeb.039123.
This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal-thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of (65)Zn(2+) by lysosomal membrane vesicles (LMV) incubated in 1 mmol l(-1) glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l(-1) alpha-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l(-1) GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal (65)Zn(2+) influx as a function of zinc concentration, in vesicles containing 1 mmol l(-1) AKG and incubated in 1 mmol l(-1) GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.
本研究描述了龙虾（Homarus americanus）肝胰腺上皮溶酶体积累和隔离细胞质中重金属的膜转运机制，从而有助于调节这些离子从饮食成分进入动物体内。本研究扩展了先前描述溶酶体通过与质子进行阳离子交换吸收金属的工作，并表明第二个平行的溶酶体转运过程涉及金属-巯基缀合物可能与阳离子反向转运体协同工作，以控制细胞质中的金属浓度。在 1 mmol l(-1) 谷胱甘肽 (GSH) 孵育的溶酶体膜囊泡 (LMV) 中，(65)Zn(2+) 的转运与没有三肽时的金属转运没有显著差异。然而，在含有 1 mmol l(-1) α-酮戊二酸 (AKG) 的培养基中预加载 LMV，然后在含有 1 mmol l(-1) GSH 的培养基中孵育，与用氯化物平衡或具有外向氯化物梯度的囊泡相比，金属摄取增加了一倍以上。在含有 1 mmol l(-1) AKG 的 LMV 中，作为锌浓度函数的溶酶体 (65)Zn(2+) 内流的动力学分析，揭示了存在一种具有低亲和力、高容量载体的金属转运体进入细胞器。这些数据表明在龙虾溶酶体膜中可能存在一种有机阴离子交换器。用兔抗大鼠 OAT1 抗体对 LMV 进行的 Western blot 分析显示，这些膜中存在一种同源的 OAT1 样蛋白（约 80 kDa）信号。这些结果和以前发表的结果表明，肝胰腺膜上存在两种金属转运体，一种是高亲和力、低容量的阳离子反向转运体，另一种是低亲和力、高容量的有机阴离子交换体。这两个系统一起具有在广泛浓度范围内调节细胞质金属的潜力。
