Capo J A, Mandal P K, Eyyunni S, Ahearn G A
Department of Biology, University of North Florida, 4567 St. Johns Bluff Road S., Jacksonville, FL 32224, USA.
J Comp Physiol B. 2005 Jan;175(1):13-20. doi: 10.1007/s00360-004-0457-x. Epub 2004 Nov 23.
The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether (65)Zn(2+) transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10-15 s and approached equilibrium by 120 s. In the absence of sodium, (65)Zn(2+) influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 microM ATP (increase in K(m) and J(max)) and inhibited by the simultaneous presence of 150 micromol l(-1) ATP+250 micromol l(-1) vanadate (decrease in both K(m) and J(max)). In the absence of ATP, (65)Zn(2+) influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l(-1)) and exhibited a Hill Coefficient of 4.03+/-1.14, consistent with the exchange of 3 Na(+)/1Zn(2+). Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle (65)Zn(2+) influx by both the ATP-dependent (K(i)=205 nmol l(-1) Ca(2+)) and sodium-dependent (K(i)=2.47 micromol l(-1) Ca(2+)) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.
龙虾（美洲螯龙虾）肝胰腺上皮细胞基底外侧细胞膜拥有三种在细胞质和血淋巴之间转运钙的转运蛋白：一种ATP依赖性钙ATP酶、一种钠钙交换体和一种对维拉帕米敏感的阳离子通道。我们使用标准离心方法制备纯化的肝胰腺基底外侧膜囊泡，并采用快速过滤程序来研究(65)Zn(2+)是否通过上述任何一种钙转运蛋白跨这种上皮细胞边界进行转运。基底外侧膜囊泡具有渗透活性，其摄取过程呈时间进程，在10 - 15秒内呈线性，120秒时接近平衡。在无钠情况下，(65)Zn(2+)内流是外部锌浓度的双曲线函数，并遵循载体转运的米氏方程。添加150微摩尔ATP可刺激这种载体转运（米氏常数K(m)和最大转运速率J(max)增加），而同时存在150微摩尔/升ATP + 250微摩尔/升钒酸盐则抑制该转运（K(m)和J(max)均降低）。在无ATP情况下，(65)Zn(2+)内流是预加载囊泡钠浓度（0、5、10、20、30、45和75毫摩尔/升）的S形函数，希尔系数为4.03±1.14，与3个Na(+)/1个Zn(2+)的交换一致。使用狄克逊分析表明，钙是ATP依赖性（抑制常数K(i)=205纳摩尔/升Ca(2+)）和钠依赖性（K(i)=2.47微摩尔/升Ca(2+)）转运过程中基底外侧膜囊泡(65)Zn(2+)内流的竞争性抑制剂。这些结果表明，锌跨龙虾肝胰腺基底外侧膜的转运主要通过ATP依赖性钙ATP酶和钠钙交换体载体蛋白进行。
