Characterization of cobalamin receptor sites in brush-border plasma membranes of the tapeworm Spirometra mansonoides.
The interaction of receptor sites in microtriches (brush-border) membranes isolated from the sparganum (larva) of the cestode (tapeworm) Spirometra mansonoides with [57Co]cyanocobalamin (CN-[57Co]Cbl) has been characterized on the basis of the following criteria: time dependence, reversibility, high-affinity binding, finite binding capacity, and ligand specificity. The association of CN-[57Co]Cbl to isolated microtriches membranes at 22 degrees C reached equilibrium in approximately 90 min. The initial rate of association follows second order kinetics with an association rate constant (ka) of 1.1 X 10(6) M-1 S-1. In the presence of an excess of unlabeled CN-Cbl, the receptor CN-[57Co]Cbl complex dissociated according to first order kinetics with a dissociation constant (kd) of 4.4 X 10(-4) S-1. An equilibrium dissociation constant (KD) of 0.4 nM was calculated on the basis of rate constants (KD = kd/ka). Scatchard analysis of equilibrium-binding data revealed a single class of high affinity binding sites with a KD of 0.3 nM and a maximum binding capacity for CN-Cbl of 221 fmol/mg of membrane protein. The stereo-specificity of CN-Cbl binding to receptor sites in isolated microtriches membranes was determined using various Cbl analogs. Receptor-site binding affinity was lowered by the following modifications: deamination of the b-, d-, and e-propionamide side chains to the corresponding monocarboxylate derivatives; modification of the B pyrrole ring to lactam or lactone; substitution of the benzimidazole moiety by a purine; modification of the benzimidazole; or deletion of the entire nucleotide moiety. On the basis of the action of trypsin and pronase, this CN-Cbl receptor may contain a relatively exposed membrane protein. The physiological role of this CN-Cbl receptor in the binding, internalization, and metabolism of Cbl is discussed in relation to cestodes and their mammalian hosts.
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D011402:Pronase / Q000494:pharmacology; D013146:Spirometra / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D014805:Vitamin B 12 / Q000378:metabolism*
D014357:Trypsin; D011402:Pronase; D014805:Vitamin B 12