Maenz D D, Chenu C, Bellemare F, Berteloot A
Department of Physiology, Faculty of Medicine, Université de Montréal, Canada.
Biochim Biophys Acta. 1991 Nov 4;1069(2):250-8. doi: 10.1016/0005-2736(91)90132-r.
The initial rates of Na(+)-dependent D-aspartate and D-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggesting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6-24 h from resuspension, however this final 'next day' uptake activity was too low to be of practical use in kinetic studies. Freezing aliquots of rabbit jejunal vesicles in liquid N2 until the time of assay resulted in complete stabilization of D-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the 'next day' stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the 'next day' specific activities of D-glucose uptake in rat jejunal brush border vesicles and D-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na(+)-dependent D-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N2 or the modified media and allowed to stabilize overnight, revealed a single transport system with a Km of 0.31-0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of D-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.
通过标准二价阳离子沉淀程序从兔和大鼠空肠分离的刷状缘膜囊泡重悬后，依赖于钠离子的D - 天冬氨酸和D - 葡萄糖摄取的初始速率显示出下降。然而，前者比后者更稳定，并且与囊泡内体积的减少密切相关，这表明转运活性的丧失可能涉及囊泡的非特异性开放以及转运蛋白的直接或间接特异性失活。两种底物的摄取速率在重悬后6 - 24小时确实趋于稳定，然而最终的“第二天”摄取活性太低，无法用于动力学研究。将兔空肠囊泡的等分试样在液氮中冷冻直至测定时，导致D - 葡萄糖摄取完全稳定。一种经过改良的匀浆缓冲液，旨在抑制广泛的磷脂酶活性，导致兔空肠囊泡的葡萄糖转运部分稳定，与未冷冻的囊泡相比，“第二天”稳定的特异性摄取活性平均提高了6倍以上。改良的匀浆缓冲液还提高了大鼠空肠刷状缘囊泡中D - 葡萄糖摄取以及兔空肠囊泡中D - 天冬氨酸摄取的稳定性和“第二天”特异性活性。它还完全稳定了后一种制剂中的囊泡内体积。对从标准匀浆培养基制备并在液氮中冷冻或改良培养基制备并过夜稳定的兔囊泡中依赖钠离子的D - 葡萄糖转运的动力学参数进行评估，发现一个Km为0.31 - 0.32 mM的单一转运系统是最适合数据的模型。因此，对匀浆培养基的改良似乎不会影响膜中D - 葡萄糖转运的功能特性。虽然在稳定囊泡方面不如快速冷冻方案有效，但结果表明，在处理像胆碱这样缓慢渗透的离子时，改良的匀浆应该是首选，因为在这种情况下，它是可靠测量通过稳定且平衡的囊泡制剂的摄取速率的唯一替代方法。
