Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor.
Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.
Amthauer
R
R
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. ramthaue@uach.cl
Tobar
L
L
Molina
H
H
Concha
M
M
Villanueva
J
J
D000818:Animals; D015153:Blotting, Western; D002347:Carps; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D011485:Protein Binding
D006735:Horseradish Peroxidase