Gerencser G A, Lee S H
Am J Physiol. 1985 Feb;248(2 Pt 2):R241-8. doi: 10.1152/ajpregu.1985.248.2.R241.
The serosa negative transepithelial potential difference across Aplysia intestine is generated by a Na+-independent, active electrogenic Cl- absorptive mechanism. In an attempt to clarify the Cl- absorptive mechanism an anion-stimulated ATPase was prepared from plasma membranes from Aplysia enterocytes utilizing differential centrifugation and sucrose density gradient techniques. ATPase activity, which could be activated by either Cl- or HCO3-, was found in the plasma membrane fraction. Maximal anion-ATPase activity was achieved with either 25 mM Cl- or 25 mM HCO3-. The apparent Km for Cl- activation of the ATPase was 10.3 mM, whereas apparent Km for HCO3- was 9.7 mM. ATP was the most effective nucleotide substrate for both HCO3- and Cl- -ATPase activities, whereas optimum pH for both activities was 7.8. These enzyme activities were inhibited more than 30% by thiocyanate (10 mM). Acetazolamide and vanadate were also found to strongly inhibit both Cl- and HCO3- -ATPase activities, whereas 10 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 1 mM furosemide, or 1 mM ouabain had little or no effect. These results are consistent with the hypothesis that the active Cl- transport mechanism in Aplysia intestine could be a Cl- -HCO3- -stimulated ATPase found in the enterocyte plasma membrane.
海兔肠道跨上皮的浆膜阴性电位差是由一种不依赖钠离子的、主动的电生性氯离子吸收机制产生的。为了阐明氯离子吸收机制，利用差速离心和蔗糖密度梯度技术从海兔肠上皮细胞的质膜中制备了一种阴离子刺激的ATP酶。在质膜部分发现了可被氯离子或碳酸氢根激活的ATP酶活性。用25 mM氯离子或25 mM碳酸氢根可实现最大阴离子ATP酶活性。ATP酶被氯离子激活的表观Km为10.3 mM，而被碳酸氢根激活的表观Km为9.7 mM。ATP是碳酸氢根和氯离子ATP酶活性最有效的核苷酸底物，而两种活性的最适pH均为7.8。这些酶活性被硫氰酸盐（10 mM）抑制超过30%。还发现乙酰唑胺和钒酸盐强烈抑制氯离子和碳酸氢根ATP酶活性，而10 microM 4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸、1 mM速尿或1 mM哇巴因几乎没有影响。这些结果与以下假设一致：海兔肠道中的主动氯离子转运机制可能是肠上皮细胞质膜中发现的一种氯离子-碳酸氢根刺激的ATP酶。
