McAbee D D, Lear M C, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
J Cell Biochem. 1991 Jan;45(1):59-68. doi: 10.1002/jcb.240450113.
We studied the effects of low temperature (20-37 degrees C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37 degrees C, hepatocytes were incubated at 37 degrees C, 31 degrees C, 25 degrees C, or 20 degrees C or treated with or without inhibitors at 37 degrees C in the absence of ligand. The cells were then assayed at 4 degrees C for 125I-asialo-orosomucoid binding, to measure receptor activity, or 125I-anti-Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin-permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub-37 degrees C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50-65% of their surface Gal receptor activity but only less than or equal to 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and 75%, respectively, were present intracellularly in monensin- and chloroquine-treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.
我们研究了低温（20 - 37摄氏度）、莫能菌素、氯喹和微管药物对分离的大鼠肝细胞中半乳糖基（Gal）受体的细胞分布及活性的影响。在37摄氏度平衡后，肝细胞于37摄氏度、31摄氏度、25摄氏度或20摄氏度孵育，或在无配体存在的情况下于37摄氏度用或不用抑制剂处理。然后在4摄氏度测定细胞与125I - 去唾液酸血清类黏蛋白的结合，以测量受体活性，或与125I - 抗Gal受体IgG的结合，以测量受体蛋白。分别在完整细胞或洋地黄皂苷通透的细胞上测量表面或总（表面和细胞内）Gal受体活性及蛋白。这些抑制剂分为两类。I型抑制剂（低于37摄氏度的温度或秋水仙碱）诱导受体重新分布但不使其失活。处理后的细胞表面Gal受体活性和蛋白最多损失40%。丢失的表面受体在细胞内重新出现，且受体活性无损失。II型抑制剂（莫能菌素或氯喹）诱导受体失活但不使其重新分布。处理后的细胞表面Gal受体活性损失50 - 65%，但其表面受体蛋白仅损失小于或等于15%。这些细胞总细胞Gal受体活性最多损失60%，而总受体蛋白无损失。在莫能菌素和氯喹处理的细胞中，分别有高达50%和75%的无活性Gal受体存在于细胞内。配体与通透处理细胞结合的丧失并非由于受体亲和力的改变。第三类，III型抑制剂（消耗ATP的代谢能量毒物）诱导Gal受体重新分布和失活（《生物化学》27:2061, 1988）。我们得出结论，肝细胞上先前已鉴定的两类Gal受体亚群中，只有一类被称为状态2受体（《生物化学杂志》265:629, 1990）的会持续循环。状态2而非状态1的Gal受体的活性和分布受这些特定药物或处理的影响不同。
