Lagarde A E, Stoeber F R
Eur J Biochem. 1975 Jul 1;55(2):343-54. doi: 10.1111/j.1432-1033.1975.tb02168.x.
Experiments were devised to test the plausibility and the predictions of a efflux rate equation which was previously derived [10]9 1. 2-Keto-3-deoxy-D-gluconate transport system conforms with universal laws relating zero-trans influx, influx at steady-state, steady-state levels of accumulation to external and internal substrate concentrations. 2. Full-time-course uptake kinetics fit the linearized graphical representation that can be inferred from the integrated rate equation. 3. Influx does not depend upon internal substrate concentration nor upon energy-coupling. 4. Zero-trans outflux (leak inot empty medium) is a first-order process (rate constant: 0.02 min-1) and not mediated by the carrier. Absence of cis-competition with D-glucuronate is in agreement with a simple diffusion mechanism. 5. Outflux increases when external substrate concentration is raised (counterflow). Outflux at steady-state equilibrates influx, and is a first-order process (rate constant: 0.15 min-1). 6. Uncoupling with azide leads to accelerate zero-trans outflux by a factor of 2-3. No further acceleration is obtained when other classical uncouplers are used. The process remains first-order, independent of the amount of carrier, and is accelerated by the presence of internal D-glucuronate as a result from trans-inhibition of the recapture. 7. Exchange outflux is all the more accelerated by azide as the carrier is less saturated. The process is clearly carrier-mediated and the outflux rate obeys a Michaelis law with respect to internal concentration. V is equal to V for influx. 8. Homo and hetero-overshoot experiments are in agreement with the participation of the carrier for mediating influx as well as outflux. 9. The kinetics of D-glucuronate outflux in a strain lacking the specific hexuronate permease but carrying the 2-keto-3-deoxy-D-glucuronate permease are similar to those obtained with 2-keto-3-deoxy-D-gluconate. We draw the conclusion that energy-coupling promotes the adjustment of outflux without interfering with influx rate. It apparently acts by reducing, in a continuous range, the affinity of the carrier facing inwards. The discussion is focused on the comparison with previously published models and on possible molecular mechanisms.
设计了实验来检验先前推导的[10]9 1. 2-酮-3-脱氧-D-葡萄糖酸盐转运系统符合将零转运流入、稳态流入、稳态积累水平与外部和内部底物浓度相关联的普遍规律。2. 全时程摄取动力学符合从积分速率方程推导得出的线性化图形表示。3. 流入不依赖于内部底物浓度，也不依赖于能量偶联。4. 零转运流出（漏入空培养基）是一级过程（速率常数：0.02分钟-1），且不由载体介导。与D-葡萄糖醛酸盐不存在顺式竞争与简单扩散机制一致。5. 当外部底物浓度升高时流出增加（逆流）。稳态时的流出与流入平衡，且是一级过程（速率常数：0.15分钟-1）。6. 用叠氮化物解偶联导致零转运流出加速2至3倍。使用其他经典解偶联剂时未获得进一步加速。该过程保持一级，与载体量无关，并且由于再捕获的反式抑制，内部D-葡萄糖醛酸盐的存在会加速该过程。7. 随着载体饱和度降低，叠氮化物对交换流出的加速作用越大。该过程显然由载体介导，并且流出速率相对于内部浓度服从米氏定律。V等于流入的V。8. 同向和异向过冲实验与载体参与介导流入和流出一致。9. 在缺乏特定己糖醛酸盐通透酶但携带2-酮-3-脱氧-D-葡萄糖酸盐通透酶的菌株中，D-葡萄糖醛酸盐流出的动力学与用2-酮-3-脱氧-D-葡萄糖酸盐获得的动力学相似。我们得出结论，能量偶联促进流出的调节而不干扰流入速率。它显然通过在连续范围内降低载体向内的亲和力起作用。讨论集中在与先前发表的模型的比较以及可能的分子机制上。
