RENATURATION AND LIGAND BLOTTING OF THE MAJOR SUBUNIT OF THE RAT ASIALOGLYCOPROTEIN RECEPTOR AFTER DENATURING POLYACRYLAMIDE-GEL ELECTROPHORESIS

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) bind terminal clust ered galactosyl or N-acetylgalactosaminyl residues with high affinity. The affinity-purified ASGP-R consists of three subunits designated RH L1, 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 nitrocellulo se, and incubation with I-125-asialo-orosomucoid (ASOR). No ligand-bin ding to any subunits could be detected when proteins such as BSA, case in, gelatin, or fat-free dry milk were used as blocking agents. Howeve r, subsequent incubation of BSA-blocked nitrocellulose blots with some nonionic detergents resulted in renaturation of RHL1. I-125-ASOR-bind ing to RHL2 or RHL3 was weaker and could be detected only after longer exposure. Similarly, direct use of detergents such as Tween 20, Nonid et P-40, or Triton X-100 as blocking agents also preserved the ASOR-bi nding activity of RHL1. Ionic detergents tested did not show any abili ty to renature the ligand-binding activity of RHL subunits. Among noni onic detergents tested, Tween 20, Tween 85, Lubrol PX, Nonidet P-40, a nd Triton X-100 were more effective than Tween 40, Tween 65, Tween 80, or Brij 35, whereas SPAN, digitonin, or octyl-glucoside showed no eff ect. Weak I-125-ASOR binding to RHL2 or RHL3 could be detected only wh en the Tween series or Lubrol PX were used. Incubation of blots with d ithiothreitol caused a dose-dependent loss of binding activity. The ca rbohydrate 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 bl otting. I-125-ASOR binding to electroblotted CRD after SDS-PAGE was al so 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 cytoplasmi c, transmembrane, or stalk domains of this subunit.