Silva Elias C C, Masui Douglas C, Furriel Rosa P, McNamara John C, Barrabin Hector, Scofano Helena M, Perales Jonas, Teixeira-Ferreira André, Leone Francisco A, Fontes Carlos Frederico L
Instituto de Bioquímica Médica, Laboratório de Estrutura e Regulação de Proteínas e ATPases, Programa de Biologia Estrutural, RJ, Brazil.
Biochim Biophys Acta. 2012 Nov;1818(11):2588-97. doi: 10.1016/j.bbamem.2012.05.009. Epub 2012 May 12.
This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.
本研究通过哺乳动物（兔）FXYD2肽特异性抗体（γC(33)）和基质辅助激光解吸电离飞行时间串联质谱技术，揭示了在蓝蟹（Callinectes danae）后鳃的微粒体Na,K-ATP酶制剂中存在FXYD2蛋白。这是首次证明无脊椎动物中存在FXYD2蛋白。向蟹鳃微粒体组分中添加外源猪FXYD2肽，以剂量依赖方式刺激了Na,K-ATP酶活性。外源猪FXYD2还显著增加了酶对K(+)、ATP和NH(4)(+)的亲和力。对Na(+)的半最大激活浓度（K(0.5)）未受影响。外源猪FXYD2使Na(+)、K(+)和ATP刺激鳃Na,K-ATP酶活性的最大反应速度（V(max)）提高了30%至40%。蟹鳃FXYD2可被蛋白激酶A（PKA）磷酸化，提示其具有与哺乳动物酶类似的调节功能。PKA磷酸化的猪FXYD2肽刺激蟹鳃Na,K-ATP酶活性的幅度达80%，约为未磷酸化肽的2倍。蛋白激酶C（PKC）磷酸化的猪FXYD2肽的刺激作用最小。这些发现证实了蟹鳃Na,K-ATP酶中存在FXYD2肽，并表明该肽在调节酶活性中起重要作用。
