Carey D J, Sommers L W, Hirschberg C B
Cell. 1980 Mar;19(3):597-605. doi: 10.1016/s0092-8674(80)80036-5.
We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.
我们提供了三条证据，证明在用³H-NeuNAc孵育的肝切片中存在微粒体内CMP-NeuNAc池：第一，相对于CMP-¹⁴C-NeuNAc（后者作为被困在囊泡之间并吸附在囊泡上的糖核苷酸的标记物添加），微粒体部分中CMP-³H-NeuNAc相对于细胞匀浆有富集；第二，在能去除所有CMP-¹⁴C-NeuNAc的条件下，通过等渗蔗糖洗涤仅能从微粒体中去除体内合成的CMP-³H-NeuNAc的一半；第三，通过低渗休克或用低浓度去污剂处理微粒体后，两种糖核苷酸完全丧失。体外将CMP-NeuNAc与微粒体一起孵育表明完整的糖核苷酸正在穿透囊泡。当糖核苷酸在糖部分被标记时，微粒体囊泡积累可溶性放射性标记产物。这种积累是可饱和的，并且依赖于时间、温度和糖核苷酸浓度，还受到底物类似物以及用蛋白酶对微粒体进行预处理的抑制。总之，这些结果为微粒体中存在CMP-NeuNAc的特定转运系统提供了有力证据。
