Mandal Prabir K, Mandal Anita, Ahearn Gregory A
Department of Biology, University of North Florida, Jacksonville, Florida 32224, USA.
J Exp Zool A Comp Exp Biol. 2006 Mar 1;305(3):203-14. doi: 10.1002/jez.a.246.
In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.
在甲壳类动物中，肝胰腺是负责重金属解毒的主要器官系统，在这个结构中，溶酶体和内质网是通过选择性隔离过程调节细胞质金属浓度的两个细胞器。本研究对负责锌摄入肝胰腺溶酶体膜囊泡（LMV）的转运过程以及该金属与相同制剂中钙、铜和镉转运之间的相互作用进行了表征。采用标准离心方法制备纯化的肝胰腺LMV，并采用快速过滤程序定量65Zn2+跨该细胞器膜的转运。LMV具有渗透反应性，其摄取时间进程在15 - 30秒内呈线性，300秒时接近平衡。65Zn2+内流是外部锌浓度的双曲线函数，遵循载体转运的米氏动力学（Km = 32.3±10.8 microM；Jmax = 20.7±2.6 pmol/mg蛋白×秒）。添加1 mM ATP可刺激这种载体转运（Km = 35.89±10.58 microM；Jmax = 31.94±3.72 pmol/mg蛋白/秒），同时存在1 mM ATP + 250 microM钒酸盐时，被明显的缓慢扩散过程所取代。毒胡萝卜素（10 microM）也是锌内流的显著抑制剂（Km = 72.87±42.75 microM；Jmax = 22.86±4.03 pmol/mg蛋白/秒），但在这方面不如钒酸盐有效。通过狄克逊分析，镉和铜被证明是通过ATP依赖的转运过程对溶酶体膜囊泡65Zn2+内流的竞争性抑制剂（镉Ki = 68.1±3.2 microM；铜Ki = 32.7±1.9 microM）。在没有ATP的情况下，外向的H+梯度刺激65Zn2+摄取，而相反方向的质子梯度抑制金属内流。本研究表明，65Zn2+通过肝胰腺溶酶体囊泡通过ATP依赖、钒酸盐、毒胡萝卜素和二价阳离子抑制的载体过程进行转运，该过程呈现米氏内流动力学，并受到外向质子梯度的刺激。这些转运特性总体表明，该转运体可能是内质网肌质内质网钙ATP酶的溶酶体同工型。
