Monoclonal antibodies as probes for the transmembrane structure of neutral endopeptidase 24.11 ('enkephalinase').
The neutral endopeptidase (EC 3.4.24.11) ('enkephalinase') is a membrane-bound metalloendopeptidase that is present in large amounts in the microvilli of the kidney proximal tubules. By immunizing mice with purified rabbit kidney brush-border membranes, we have obtained four different monoclonal antibodies that recognize this enzyme in dot-blot and Western-blot assays and can be used for immunoprecipitation of neutral endopeptidase from crude kidney solubilizates. One of these monoclonal antibodies (2B12) allows the labeling of proximal tubule cells with colloidal gold particles. This monoclonal antibody also binds to native brush-border membrane vesicles (which are mostly in the right-side-out configuration) and recognizes an epitope which is destroyed after reduction and alkylation of the protein. By contrast, all three other monoclonal antibodies (21G10, 23B11 and 22E2) compete for another epitope of neutral endopeptidase that is not exposed at the extracytoplasmic surface either in intact cells or in sealed brush-border vesicles. Permeabilization of the vesicles with digitonin, however, restores the full binding activity. Binding of these antibodies is not altered by prior reduction and alkylation of the protein. Taken together, these results strongly suggest that the 2B12 monoclonal antibody binds a conformational epitope located on the ectodomain of the enzyme, whereas the three others (21G10, 23B11 and 22E2) bind to a common or to overlapping epitopes located on the cytosolic domain. These results also demonstrate unambiguously the transmembrane nature of neutral endopeptidase.
Aubry
M
M
Départment de Biochimie, Université de Montréal, Canada.
Zollinger
M
M
Fortin
S
S
Vénien
C
C
LeGrimellec
C
C
Crine
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D001485:Basement Membrane / Q000201:enzymology; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D007672:Kidney Cortex / Q000201:enzymology / Q000648:ultrastructure; D015260:Neprilysin / Q000276:immunology*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015260:Neprilysin