| Abstrakt: |
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) bind terminal clustered galactosyl or N-acetylgalactosaminyl residues with high affinity. The affinity-purified ASGP-R consists of three subunits designated RHL1, RHL2, and RHL3. The ligand-binding activity of individual subunits was investigated by ligand blotting, after separation of subunits by SDS-PAGE under nonreducing conditions, electrotransfer to nitrocellulose, and incubation with 125I-asialo-orosomucoid (ASOR). No ligand-binding to any subunits could be detected when proteins such as BSA, casein, gelatin, or fat-free dry milk were used as blocking agents. However, subsequent incubation of BSA-blocked nitrocellulose blots with some nonionic detergents resulted in renaturation of RHL1. 125I-ASOR-binding to RHL2 or RHL3 was weaker and could be detected only after longer exposure. Similarly, direct use of detergents such as Tween 20, Nonidet P-40, or Triton X-100 as blocking agents also preserved the ASOR-binding activity of RHL1. Ionic detergents tested did not show any ability to renature the ligand-binding activity of RHL subunits. Among nonionic detergents tested, Tween 20, Tween 85, Lubrol PX, Nonidet P-40, and Triton X-100 were more effective than Tween 40, Tween 65, Tween 80, or Brij 35, whereas SPAN, digito-nin, or octyl-glucoside showed no effect. Weak 125I-ASOR binding to RHL2 or RHL3 could be detected only when the Tween series or Lubrol PX were used. Incubation of blots with dithiothreitol caused a dose-dependent loss of binding activity. The carbohydrate recognition domain (CRD) of RHL1, isolated after subtilisin digestion of ASGP-R bound to ASOR-Sepharose, retained ligand-binding activity as assessed by its binding to ASOR-Sepharose and by ligand blotting. 125I-ASOR binding to electroblotted CRD after SDS-PAGE was also dependent on the presence of nonionic detergents. We conclude that restoration of ligand-binding activity of RHL1 after SDS-PAGE by some nonionic detergents is not dependent on the presence of the cytoplasmic, transmembrane, or stalk domains of this subunit. [ABSTRACT FROM PUBLISHER] |
