Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine.
Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine.
复制标题
肾刷状缘膜囊泡中氨基酸的转运。
DOI：
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发表时间：
1977
影响因子：
4.8
通讯作者：
B. Sacktor
中科院分区：
文献类型：
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作者：
S. Fass;M. Hammerman;B. Sacktor
利用兔肾近端小管刷状边缘的膜泡研究了l -丙氨酸的转运。肾膜与l -丙氨酸而非d -丙氨酸预孵育，加速了l -丙氨酸的交换扩散，即立体定向反运输。l -丙氨酸的平衡吸收随着介质渗透压的增加而降低。外推到无限介质渗透压，即零泡内空间，表明没有摄取。这些发现表明，l -丙氨酸的摄取代表转运到膜囊泡，而不是表面结合到膜上。外部培养培养基和囊泡内培养基之间存在Na+梯度，刺激l -丙氨酸摄取。氨基酸在囊泡中的积累在5分钟时达到最大，然后减少，表明外排。在有Na+梯度的情况下，最终的吸收水平与没有梯度的情况下的吸收水平相同，表明平衡已经建立。在“超调”的高峰期，l -丙氨酸的摄取几乎是最终平衡值的两倍。这些结果表明，一个大的泡外到泡内的Na+梯度的施加影响了l -丙氨酸进入肾刷状边界膜泡的瞬时运动，而不是其浓度梯度。l -丙氨酸摄取刺激是Na+特异性的。当囊内介质不含Na+时，吸收速率随外介质中Na+浓度的增加而增强。增加Na+梯度降低l -丙氨酸的表观Km。在没有Na+梯度的情况下，L-丙氨酸和d -丙氨酸的摄取率是相同的。在Na+梯度存在的情况下，d -丙氨酸的摄取速率受到刺激，但明显低于l -丙氨酸的摄取速率。在一定浓度的氨基酸下，l -丙氨酸的吸收反映了Na+梯度依赖性和非依赖性转运系统的贡献总和。依赖体系在约2 mM l -丙氨酸时饱和。独立系统表现出最小的饱和性，并且本身可能代表被动扩散和“载流子”介导系统的总和。在生理浓度的l -丙氨酸下，Na+梯度依赖性摄取速率是无梯度时的5倍。如果存在K+梯度（囊泡大于中等），那么缬霉素可以增强Na+梯度依赖性的l -丙氨酸摄取。这一发现表明，l -丙氨酸进入肾刷状边界膜囊的Na+梯度依赖性运输是一个电致过程，并表明膜电位是l -丙氨酸运输的决定因素。在Na+梯度存在的情况下，l -丙氨酸的摄取被其他中性l -氨基酸强烈抑制。亚胺酸和甘氨酸也有抑制作用，但酸性和碱性氨基酸没有作用。在没有Na+梯度的情况下，几乎没有选择性竞争。
The transport of L-alanine was studied using membrane vesicles derived from the brush borders of the rabbit renal proximal tubule. Preincubation of the renal membranes with L-alanine, but not D-alanine, accelerated exchange diffusion of L-alanine, i.e. stereospecific counter transport. The equilibrium uptake of L-alanine decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, i.e. zero intravesicular space, indicates no uptake. These findings demonstrate that the uptake of L-alanine represents transport into membrane vesicles and not surface binding to the membrane. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium stimulated L-alanine uptake. Accumulation of the amino acid in the vesicles was maximal at 5 min and then decreased, indicating efflux. The final level of uptake in the presence of the Na+ gradient was identical with that obtained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the "overshoot" the uptake of L-alanine was slmost twice the final equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of L-alanine into renal brush border membrane vesicles against its concentration gradient. Stimulation of L-alanine uptake was specific for Na+. When the intravesicular medium contained no Na+, the rate of uptake enhanced with increases in the concentration of Na+ in the external medium. Increasing the Na+ gradient lowered the apparent Km for L-alanine. In the absence of the Na+ gradient, the rates of uptake of L- and D-alanine were identical. In the presence of the Na+ gradient, the rate of D-alanine uptake was stimulated, but significantly less than that of L-alanine. The uptake of L-alanine, at a given concentration of amino acid reflected the sum of the contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at about 2 mM L-alanine. The independent system exhibited minimal saturability and may itself represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations of L-alanine, the rate of the Na+ gradient-dependent uptake was 5-fold that in the absence of the gradient. Valinomycin enhanced the Na+ gradient-dependent uptake of L-alanine, provided a K+ gradient (vesicle greater than medium) was present. This finding indicates that the Na+ gradient-dependent transport of L-alanine into renal brush border membrane vesicles is an electrogenic process and suggests that the membrane potential is a determinant of L-alanine transport. In the presence of a Na+ gradient, the uptake of L-alanine was strongly inhibited by other neutral L-amino-acids. Imino acids and glycine also inhibited, but acidic and basic amino acids were without effect. In the absence of a Na+ gradient, little selective competition was found...