Nakada H, Sawamura T, Tashiro Y
J Biochem. 1985 Jan;97(1):341-51. doi: 10.1093/oxfordjournals.jbchem.a135059.
Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [3H]leucine and pulse-chasing after 10 min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15 min, in the smooth microsomes and the heavy Golgi subfraction (GF3) at 25 min and in the intermediate plus light Golgi subfraction (GF1+2) at 30 min. By 60 min, the labeling in GF1+2 had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20 min, increased rapidly and also reached a constant level at 40-60 min. Intracellular movement of the newly synthesized receptor in the GF1+2 and plasma membrane fractions was also investigated by purifying the receptor protein from the GF1+2 and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120 min. The receptor of the various membrane fractions was also pulse-labeled in vivo for 20 min simultaneously with [3H]glucosamine and [14C]leucine, and pulse-chased for the following 40 min. After pulse-labeling for 20 min, the ratio of the radioactivity of [3H]glucosamine or [3H]sialic acid to [14C]leucine of the receptor from the rough and smooth microsomes, and GF3, GF2, and GF1 increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of 3H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of 14C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [14C]leucine labeled receptor to the plasma membrane. After chasing for 40 min, however, the same radioactivity ratios of the GF1 and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.
利用针对该受体的单特异性抗体，通过生物化学方法研究了新合成的去唾液酸糖蛋白受体的细胞内运输。静脉注射[3H]亮氨酸进行脉冲标记，并在10分钟后注射环己酰亚胺进行脉冲追踪，结果显示15分钟时粗面微粒体中的受体标记量最大，25分钟时滑面微粒体和高尔基体重亚组分（GF3）中的受体标记量最大，30分钟时中间高尔基体和轻高尔基体亚组分（GF1 + 2）中的受体标记量最大。到60分钟时，GF1 + 2中的标记量下降并趋于平稳。在质膜组分中，标记的受体在20分钟时首次出现，迅速增加，并在40 - 60分钟时也达到稳定水平。还通过亲和色谱从GF1 + 2和质膜组分中纯化受体蛋白，研究了新合成的受体在GF1 + 2和质膜组分中的细胞内移动。结果表明，60 - 120分钟后，两个组分中受体的比放射性达到平衡。同时用[3H]葡糖胺和[14C]亮氨酸对体内各种膜组分的受体进行20分钟的脉冲标记，并在接下来的40分钟进行脉冲追踪。脉冲标记20分钟后，粗面和滑面微粒体以及GF3、GF2和GF1中受体的[3H]葡糖胺或[3H]唾液酸与[14C]亮氨酸的放射性比值依次增加。质膜组分中受体的该比值无限高，这是因为虽然大量的3H放射性掺入高尔基体中的受体，但由于[14C]亮氨酸标记的受体到达质膜延迟，质膜中同一受体掺入的14C放射性可忽略不计。然而，追踪40分钟后，GF1和质膜组分的相同放射性比值彼此接近。所有这些结果强烈表明，新合成的受体可能通过受体蛋白在两个膜之间的反复循环，在反式高尔基体成分和质膜之间迅速达到分布平衡。
