Pardon R S, Noël F
Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro, Brazil.
Biochem Pharmacol. 1994 Jan 20;47(2):331-6. doi: 10.1016/0006-2952(94)90024-8.
Binding experiments with [3H]ouabain were performed to investigate the presence of (Na+ + K+)-ATPase (EC3.6.1.3) isoforms in adult male Schistosoma mansoni, the trematode responsible for human schistosomiasis. Non-linear regression analysis of equilibrium experiments performed with homogenates in a Mg-Pi medium indicated the presence of about 10% (Bmax = 223 +/- 67 fmol/mg protein) high-affinity sites (KD = 0.285 +/- 0.045 microM) and 90% (Bmax = 2117 +/- 348 fmol/mg protein) sites with a 20-fold lower affinity (KD = 4.9 +/- 1.28 microM). This was confirmed by their-exponential decay of [3H]ouabain dissociation. Furthermore, determination of association and dissociation rate constants indicated that the two classes of binding sites differed by their dissociation rate constants for ouabain (k-1 = 0.0185 +/- 0.0019 min-1 and 0.0997 +/- 0.0528 min-1 for high- and low-affinity sites, respectively). Surprisingly, the association rate constant measured for ouabain binding to S. mansoni homogenate (0.038 microM-1.min-1) was lower (25- to 80-fold) than the one usually observed for mammalian enzymes. This is the first direct evidence for the existence of (Na+ + K+)-ATPase isoforms in platyhelminths, invertebrates of great importance from the phylogenetic point of view.
采用[3H]哇巴因进行结合实验，以研究成年雄性曼氏血吸虫（导致人类血吸虫病的吸虫）中（Na+ + K+）-ATP酶（EC3.6.1.3）同工型的存在情况。对在Mg-Pi培养基中用匀浆进行的平衡实验进行非线性回归分析表明，存在约10%（Bmax = 223 +/- 67 fmol/mg蛋白质）的高亲和力位点（KD = 0.285 +/- 0.045 microM）和90%（Bmax = 2117 +/- 348 fmol/mg蛋白质）亲和力低20倍的位点（KD = 4.9 +/- 1.28 microM）。[3H]哇巴因解离的指数衰减证实了这一点。此外，缔合和解离速率常数的测定表明，两类结合位点的哇巴因解离速率常数不同（高亲和力和低亲和力位点的k-1分别为0.0185 +/- 0.0019 min-1和0.0997 +/- 0.0528 min-1）。令人惊讶的是，测得的哇巴因与曼氏血吸虫匀浆结合的缔合速率常数（0.038 microM-1.min-1）低于通常在哺乳动物酶中观察到的值（低25至80倍）。这是关于扁形虫中存在（Na+ + K+）-ATP酶同工型的首个直接证据，扁形虫是从系统发育角度来看非常重要的无脊椎动物。
