Sundin D P, Cohen M, Dahl R, Falk S, Molitoris B A
Department of Medicine, University of Colorado School of Medicine, Denver.
Am J Physiol. 1994 Sep;267(3 Pt 2):F380-9. doi: 10.1152/ajprenal.1994.267.3.F380.
The uptake mechanism(s) of low-molecular-weight proteins by proximal tubule cells remains incompletely characterized. We utilized a biochemical and semiquantitative morphological approach to better characterize the endocytic pathway of an anionic protein, beta 2-microglobulin (beta 2M), in the rat proximal tubule. Indirect immunogold techniques revealed beta 2M was taken up via a classic receptor-mediated endocytic pathway. In vitro biochemical and morphological characterization of iodinated beta 2M and gold-conjugated beta 2M (gold-beta 2M) binding to isolated brush-border membrane vesicles (BBMV) documented specific and quantitatively similar binding interactions of the modified beta 2M with BBMV. Kinetic characterization of the in vivo endocytic pathway of gold-beta 2M was undertaken using microinfusion of individual tubules. beta 2M initially bound at the apical surface, was internalized into subapical coated vesicles and delivered to endosomal-like structures within 5 min, and, finally, was concentrated in lysosomal-like structures within 15 min. This uptake was inhibited by excess unconjugated beta 2M. In addition, we directly showed that uptake did not occur across the basolateral surface. Finally, by passing solubilized BBMV over beta 2M affinity columns we were able to isolate binding activity.
近端小管细胞对低分子量蛋白质的摄取机制仍未完全明确。我们采用生化和半定量形态学方法，以更好地描述大鼠近端小管中阴离子蛋白β2微球蛋白（β2M）的内吞途径。间接免疫金技术显示，β2M通过经典的受体介导内吞途径被摄取。对碘化β2M和金偶联β2M（金-β2M）与分离的刷状缘膜囊泡（BBMV）结合的体外生化和形态学特征进行研究，记录了修饰后的β2M与BBMV之间特异性且在数量上相似的结合相互作用。使用单个小管微量灌注法对金-β2M体内内吞途径进行动力学特征研究。β2M最初结合在顶端表面，5分钟内被内化到顶端下的被膜小泡中，并被递送至类内体结构，最终在15分钟内聚集在类溶酶体结构中。这种摄取被过量的未偶联β2M所抑制。此外，我们直接证明摄取不会发生在基底外侧表面。最后，通过使溶解的BBMV通过β2M亲和柱，我们能够分离出结合活性。
