Masui D C, Furriel R P M, McNamara J C, Mantelatto F L M, Leone F A
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto 14040-901, SP, Brazil.
Comp Biochem Physiol C Toxicol Pharmacol. 2002 Aug;132(4):471-82. doi: 10.1016/s1532-0456(02)00110-2.
The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.
分析了钠（Na⁺）、钾（K⁺）、铵（NH₄⁺）和三磷酸腺苷（ATP）对来自蝉蟹（Callinectes danae）鳃微粒体部分的（钠，钾）-ATP酶的调节作用。ATP在高亲和力结合位点水解，最大速率为V = 35.4 ± 2.1 Umg⁻¹，半最大激活浓度（K₀.₅）= 54.0 ± 3.6 nM，遵循协同动力学（n(H)=3.6）。在低亲和力位点，该酶水解ATP遵循米氏动力学，米氏常数（Kₘ）= 55.0 ± 3.0 μM，V = 271.5 ± 17.2 Umg⁻¹。这是首次证明甲壳类动物的（钠，钾）-ATP酶具有两个ATP水解位点。钠（K₀.₅ = 5.80 ± 0.30 mM）、镁（K₀.₅ = 0.48 ± 0.02 mM）和钾离子（K₀.₅ = 1.61 ± 0.06 mM）的刺激表现出位点间相互作用，而铵离子的刺激遵循米氏动力学（Kₘ = 4.61 ± 0.27 mM）。哇巴因（Kᴵ = 147.2 ± 7.μM）和原钒酸盐（Kᴵ = 11.2 ± 0.6 μM）完全抑制ATP酶活性，表明不存在污染的ATP酶和/或中性磷酸酶活性。铵离子和钾离子协同刺激该酶，使比活性增加高达90%，表明这些离子结合到分子上的不同位点。每种离子的存在调节另一种离子对酶的刺激作用。讨论了铵离子对（钠，钾）-ATP酶活性的调节以及底栖蟹中NH₄⁺的排泄。
