Li M, Distler J J, Jourdian G W
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
Arch Biochem Biophys. 1990 Nov 15;283(1):150-7. doi: 10.1016/0003-9861(90)90625-9.
The aggregation state of low molecular weight mannose 6-phosphate receptor from bovine testis was determined in membrane preparations and in purified soluble preparations. The effect of aggregation on binding of the receptor to immobilized pentamannose 6-phosphate was also examined. Nonreducing SDS-PAGE followed by immunoblotting revealed that interchain disulfide bonds exist in detergent-solubilized and purified receptor preparations, but not in membrane-associated receptor. Reduction of the receptor with dithiothreitol abolished its ligand binding activity and drastically altered its ability to bind antibodies. The results of receptor crosslinking and molecular sieving chromatography studies suggest that the receptor exists in membranes as a noncovalently linked dimer and in solution as oligomeric forms, largely as a tetramer. The formation of the tetramer is affected by the concentration of the receptor, but not by its solubilization from membranes with detergent, nor by the presence of mannose 6-phosphate. Mono-, di-, and tetramer forms of 125I-labeled receptor were separated by molecular sieving chromatography and examined for their ability to bind to immobilized ligand, agarose-pentamannose-phosphate. The order of binding observed was tetramer greater than dimer greater than monomer. Binding of the monomer and dimer to immobilized ligand was dependent on the presence of divalent cations while the tetramer had little requirement for divalent cations.
在膜制剂和纯化的可溶性制剂中测定了来自牛睾丸的低分子量甘露糖6-磷酸受体的聚集状态。还研究了聚集对受体与固定化五甘露糖6-磷酸结合的影响。非还原SDS-PAGE后进行免疫印迹显示，在去污剂溶解和纯化的受体制剂中存在链间二硫键，但在膜相关受体中不存在。用二硫苏糖醇还原受体消除了其配体结合活性，并极大地改变了其结合抗体的能力。受体交联和分子筛色谱研究结果表明，受体在膜中以非共价连接的二聚体形式存在，在溶液中以寡聚体形式存在，主要是四聚体。四聚体的形成受受体浓度的影响，但不受其用去污剂从膜中溶解的影响，也不受甘露糖6-磷酸存在的影响。通过分子筛色谱分离了125I标记受体的单聚体、二聚体和四聚体形式，并检测了它们与固定化配体琼脂糖-五甘露糖-磷酸结合的能力。观察到的结合顺序是四聚体大于二聚体大于单体。单体和二聚体与固定化配体的结合依赖于二价阳离子的存在，而四聚体对二价阳离子的需求很少。
