Leone Francisco A, Garçon Daniela P, Lucena Malson N, Faleiros Rogério O, Azevedo Sergio V, Pinto Marcelo R, McNamara John C
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
Comp Biochem Physiol B Biochem Mol Biol. 2015 Aug;186:59-67. doi: 10.1016/j.cbpb.2015.04.010. Epub 2015 Apr 28.
We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.
我们评估了广盐性螃蟹华丽青蟹在从海水（盐度33‰）到低盐度（21‰）的10天适应期内，其第6和第7对后鳃中（Na⁺，K⁺）-ATP酶活性以及α亚基的蛋白质和基因表达。转移到21‰盐度后1小时内，（Na⁺，K⁺）-ATP酶活性下降，24小时后恢复，10天后达到最大值约180 nmol Pi min⁻¹ mg⁻¹（约增加2.5倍）。无论盐度如何，第6鳃中的（Na⁺，K⁺）-ATP酶活性比第7鳃大约高1.5倍。暴露于低盐度1至2小时内，两鳃中（Na⁺，K⁺）-ATP酶α亚基mRNA的相对表达均增加，暴露24小时后达到约8倍的最大值，在适应低盐度10天后略有下降。α亚基mRNA表达的这种增加可能是适应低盐度10天后（Na⁺，K⁺）-ATP酶活性增加的基础。与第7鳃相比，第6鳃中酶对ATP的亲和力更大，而哇巴因亲和力在第7鳃中更大。蛋白质印迹分析确定了一条针对（Na⁺，K⁺）-ATP酶α亚基的单一免疫反应条带，其Mr约为105 kDa，与鳃的编号和低盐度适应无关。尽管存在这些差异，但第6和第7鳃在从稀释介质中摄取盐分方面似乎发挥着相似的功能。获得的华丽青蟹鳃（Na⁺，K⁺）-ATP酶的部分cDNA序列（GenBank登录号KF056804）与其他梭子蟹鳃的相似序列具有97%至91%的同一性。本文讨论了适应低盐度过程中鳃（Na⁺，K⁺）-ATP酶活性的调节。
