Detergent dissociation of bovine liver phosphomannosyl binding protein.
We have reported previously the isolation and partial characterization of a 215-kilodalton (Kd) phosphomannosyl binding protein from bovine liver membranes [3,9]. In the present studies evidence is presented that the binding protein is an aggregate. Four N-terminal amino acids were detected, and the complex could be dissociated into subunits. Bovine liver membranes were extracted with the detergent, Zwittergent, in the presence of protease inhibitors. The extract was subjected to affinity chromatography on phosphomannan-Sepharose 4B, and proteins with apparent Mr values of 215 and 57 Kd were eluted with mannose 6-phosphate. As reported previously, extraction with Triton X-100 yielded only the higher molecular weight material. When the binding protein was incubated at 4 degrees C in the presence of Zwittergent TM 3-14 the 215-Kd form slowly dissociated into smaller subunits; after two months, the major species had an apparent Mr of 57 Kd. The subunits derived from the binding protein were recognized by antiserum raised against purified binding protein. Dissociation of the binding protein by Zwittergent was enhanced by incubation at 37 degrees C, the presence of dithiothreitol, and low pH values. The subunit mixture enriched in the 57-Kd subunit had a lowered ability to bind ligands containing the phosphomannosyl recognition marker. Binding was partially restored (greater than 48% of the initial value) when dissociated receptor was back exchanged with Triton X-100.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D005696:Galactosidases / Q000378:metabolism; D008099:Liver / Q000032:analysis*; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear
D000596:Amino Acids; D003902:Detergents; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C034304:cation-dependent mannose-6-phosphate receptor; D005696:Galactosidases