高盐环境下，寄居蟹 Clibanarius vittatus（十足目，异尾下目）血淋巴离子调节和鳃（Na⁺，K⁺）-ATP 酶的动力学特性。
Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity.
机构信息
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Brazil.
出版信息
Comp Biochem Physiol B Biochem Mol Biol. 2012 Apr;161(4):380-91. doi: 10.1016/j.cbpb.2012.01.003. Epub 2012 Jan 11.
We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na(+), K(+))-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg(-1) H(2)O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg(-1) H(2)O). Hemolymph [Na(+)] (323.0 ± 2.5 mmol L(-1)) and [Mg(2+)] (34.6 ± 1.0 mmol L(-1)) are hypo-regulated while [Ca(2+)] (22.5 ± 0.7 mmol L(-1)) is hyper-regulated; [K(+)] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L(-1)) but hypo-regulated (6.2 ± 0.7 mmol L(-1)) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm=46.5 ± 3.5 Umg(-1); K(0.5)=7.07 ± 0.01 μmol L(-1)) and a low-affinity ATP binding site (Vm=108.1 ± 2.5 U mg(-1); K(0.5)=0.11 ± 0.3 mmol L(-1)), both obeying cooperative kinetics, were disclosed. Modulation of (Na(+), K(+))-ATPase activity by Mg(2+), K(+) and NH(4)(+) also exhibits site-site interactions, but modulation by Na(+) shows Michaelis-Menten kinetics. (Na(+), K(+))-ATPase activity is synergistically stimulated up to 45% by NH(4)(+) plus K(+). Enzyme catalytic efficiency for variable [K(+)] and fixed [NH(4)(+)] is 10-fold greater than for variable [NH(4)(+)] and fixed [K(+)]. Ouabain inhibited ≈80% of total ATPase activity (K(I)=464.7 ± 23.2 μmol L(-1)), suggesting that ATPases other than (Na(+), K(+))-ATPase are present. While (Na(+), K(+))-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1)mg(-1)) and 45‰-acclimated crabs (around 154 nmol Pi min(-1)mg(-1)), ATP affinity decreases 110-fold and Na(+) and K(+) affinities increase 2-3-fold in 45‰-acclimated crabs.
我们研究了在适应 45‰盐度 10 天后的潮间带寄居蟹 Clibanarius vittatus 的血淋巴离子调节和鳃微粒体（Na(+), K(+))-ATPase 的动力学特性。在 45‰时，血淋巴渗透压被低调节（1102.5 ± 22.1 mOsm kg(-1) H(2)O），但与新鲜捕获的螃蟹相比升高（801.0 ± 40.1 mOsm kg(-1) H(2)O）。血淋巴 [Na(+)]（323.0 ± 2.5 mmol L(-1)) 和 [Mg(2+)]（34.6 ± 1.0 mmol L(-1)) 被低调节，而 [Ca(2+)]（22.5 ± 0.7 mmol L(-1)) 被高调节；新鲜捕获的螃蟹中 [K(+)]（17.4 ± 0.5 mmol L(-1)) 被高调节，但在 45‰时被低调节（6.2 ± 0.7 mmol L(-1))。尽管 Western blot 分析显示只有一个免疫反应性条带，但在 45‰适应的螃蟹中蛋白表达模式发生了改变，这表明存在一种单一的（Na(+), K(+))-ATPase α-亚基同工酶，分布在不同密度的膜部分。揭示了一种高亲和力（Vm=46.5 ± 3.5 Umg(-1); K(0.5)=7.07 ± 0.01 μmol L(-1)) 和低亲和力 ATP 结合位点（Vm=108.1 ± 2.5 U mg(-1); K(0.5)=0.11 ± 0.3 mmol L(-1))，均服从协同动力学。（Na(+), K(+))-ATPase 活性被 Mg(2+)、K(+)和 NH(4)(+) 的调节也表现出位点-位点相互作用，但 Na(+)的调节显示出米氏动力学。（Na(+), K(+))-ATPase 活性通过 NH(4)(+) 加 K(+)协同刺激高达 45%。对于可变 [K(+)]和固定 [NH(4)(+)]，酶的催化效率比可变 [NH(4)(+)]和固定 [K(+)]高 10 倍。哇巴因抑制约 80%的总 ATPase 活性（K(I)=464.7 ± 23.2 μmol L(-1))，表明存在除（Na(+), K(+))-ATPase 之外的其他 ATP 酶。虽然在新鲜捕获的螃蟹中（约 142 nmol Pi min(-1)mg(-1))和适应 45‰的螃蟹中（约 154 nmol Pi min(-1)mg(-1))的（Na(+), K(+))-ATPase 活性相似，但在适应 45‰的螃蟹中，ATP 亲和力降低 110 倍，Na(+)和 K(+)亲和力增加 2-3 倍。
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