Gross S K, Lyerla T A, Williams M A, McCluer R H
E.K. Shriver Center for Mental Retardation, Waltham, MA 02254.
Mol Cell Biochem. 1992 Dec 2;118(1):61-6. doi: 10.1007/BF00249695.
In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bgJ/bgJ mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bgJ/bgJ beige mutants were grown in D-valine medium and glycosphingolipids labeled with [3H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [3H]palmitate into glycosphingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes.
在正常的C57BL/6J雄性小鼠中，肾脏糖鞘脂的一个特定亚群与溶酶体来源的多层小体相关，约占肾脏总糖脂的10%，每天都会排泄到尿液中。这种排泄在该品系的bgJ/bgJ突变体小鼠的肾脏中被阻断，糖鞘脂在肾脏中积累。为了在体外研究这一过程，在米色小鼠肾细胞培养物中研究了糖鞘脂的代谢和排泄。来自雄性C57BL/6J对照小鼠和bgJ/bgJ米色突变体小鼠的原代肾细胞培养物在D-缬氨酸培养基中生长，并用[3H]棕榈酸标记糖鞘脂。正如我们之前所表明的，米色肾细胞培养物中维持着形态改变的巨大溶酶体。米色-J细胞和对照细胞合成相同类型的糖鞘脂，但通过高效液相色谱法测定，突变体细胞中这些化合物的含量在数量上高于对照细胞。以每分钟计数/毫克蛋白质为基础，米色-J细胞比对照细胞将更多的[3H]棕榈酸掺入糖鞘脂中，并且米色细胞中的比活性（每分钟计数/皮摩尔糖鞘脂）较低。在24小时内，来自米色-J细胞的培养基中积累的糖鞘脂比来自对照细胞的培养基中更多。通过高效液相色谱法测定，释放到培养基中的糖鞘脂主要是非溶酶体类型，对照细胞和突变体细胞都保留了与体内排泄的溶酶体多层小体相关的糖鞘脂。因此，米色细胞原代培养物中溶酶体糖鞘脂水平的升高和形态异常的溶酶体并不是由溶酶体分泌的突变阻断引起的。
