Berteloot A, Malo C, Breton S, Brunette M
Department of Physiology, Faculty of Medicine, University of Montréal, Canada.
J Membr Biol. 1991 Jun;122(2):111-25. doi: 10.1007/BF01872635.
Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10-40 microliters) with an incubation medium (0.2-1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4 sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15-20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na(+)-D-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.
（刷状缘）膜囊泡中的动力学数据依赖于初始速率假设的有效性来进行解释，并且其实验测量依赖于使用快速过滤技术的示踪剂通量研究。由于缺乏能够进行实时数据分析的技术，这些数据在一定程度上受到了限制。在本文中，我们报告了我们成功设计的一种快速采样、快速过滤装置（FSRFA），该装置似乎填补了这一技术空白，具有以下特点：（i）将少量囊泡（10 - 40微升）与孵育介质（0.2 - 1.0毫升）进行快速注射（5毫秒）和混合（少于100毫秒）；（ii）根据预定的时间安排（0.25至9999秒的任何时间组合），对摄取混合物进行快速（根据样品体积为20至80毫秒）和多次（以最大速率4秒最多可采集18个样品）采样，随后在终止溶液中快速淬灭（约5毫秒）；（iii）对淬灭后的摄取介质进行快速、自动且与采样同步的过滤和洗涤（第一次过滤仅需15 - 20秒，随后进行两次洗涤和额外过滤）。以成人人类空肠刷状缘膜囊泡和钠 - D - 葡萄糖共转运作为模型进行的实验表明，FSRFA能够准确再现快速过滤技术的人工操作部分，同时允许非常精确地测定初始速率。此外，FSRFA的设计还尽可能提供了多功能性，在其当前版本中，可以非常精确地控制孵育温度，并且还允许执行一些外排实验方案。最后，其模块化设计将快速采样单元与快速过滤装置分开，这应该有助于将其应用扩展到运输测量以外的其他领域。
