The aggregation and dissociation properties of a low molecular weight mannose 6-phosphate receptor from bovine testis.
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.
Li
M
M
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
Distler
J J
JJ
Jourdian
G W
GW
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism*; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
D007106:Immune Sera; D046911:Macromolecular Substances; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D004229:Dithiothreitol