Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys - hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 - were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes.

译文

非洲爪蟾卵母细胞是研究膜蛋白功能的有价值的工具。然而,世界各地的法规,特别是巴西的法规,使得非洲爪蟾青蛙的进口不切实际,在某些情况下是不可能的。在这里,作为替代方案,我们评估了在巴西市售的北美水生牛蛙Lithobates catesebeianus对水通道蛋白 (AQP) 蛋白异源表达的有用性。我们已经开发了一种结合了简短的胶原酶治疗和机械去卵泡术的方法,用于从litobates卵巢中分离单个卵母细胞。我们发现它们的大小,形状和外观与非洲爪蟾卵母细胞相似,并且在注射cRNA或水后可以耐受并存活。此外,表面生物素化,蛋白质印迹分析和渗透水渗透率 (Pf) 的测量表明,岩质卵母细胞可以在质膜上表达AQPs,并显着增加卵母细胞的Pf。实际上,Pf值与从非洲爪蟾卵母细胞收集的历史值相似。由于水通道的喉咙中存在对汞敏感的半胱氨酸 (Cys或C),表达人 (h) AQP1,hAQP1FLAG [FLAG,短蛋白标签 (DYKDDDDK) 的卵母细胞的Pf添加到AQP1的N端],hAQP8,并且大鼠 (r) AQP9被汞化合物对氯代苯磺酸盐 (pCMBS) 抑制,而缺少该Cys-hAQP1C189S突变体的aqp [残基Cys 189被丝氨酸 (Ser或S) 取代] 和hAQP7-对汞不敏感。与先前对非洲爪蟾卵母细胞的研究相反,rAQP3也被发现对汞不敏感,这与汞敏感的Cys (Cys 11) 位于细胞内是一致的。因此,我们认为,对于目前无法获得非洲爪蟾卵母细胞的国际研究人员来说,这些卵母细胞是一种易于获得的膜蛋白功能表达和研究系统。

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