AJP - Regulatory, Integrative and Comparative Physiology, Vol 264, Issue 4 804-R810, Copyright © 1993 by American Physiological Society
Electrogenic 2 Na/1 H antiport in crustacean epithelium is inhibited by a monoclonal antibody
H. Gert de Couet, L. Busquets-Turner, A. Gresham and G. A. Ahearn
Department of Zoology, University of Hawaii at Manoa, Honolulu 96822.
We have previously published evidence that suggests that Na/H exchange in
crustacean and echinoderm epithelia occurs by an electrogenic antiporter
protein with two external cation binding sites that accommodate Na,
amiloride, or Ca and display a 2:1 monovalent cation antiport
stoichiometry. The present study is an initial investigation into the
molecular biology of this invertebrate electrogenic exchanger to ascertain
its structural similarity to the analogous vertebrate electroneutral
antiport system. A panel of monoclonal antibodies was prepared against
components of lobster hepatopancreatic epithelial brush-border membranes
and assayed immunohistochemically and by Western blotting. The antibodies
were tested further in functional assays for their ability to interfere
with electrogenic 2 Na/1 H antiport in isolated hepatopancreatic
brush-border membrane vesicles. One cell line was identified producing an
antibody that significantly inhibited the electrogenic exchange of cations
by these membrane preparations and recognized a single protein band on
Western blots of hepatopancreas, antennal gland, and gill epithelia
corresponding to a molecular mass of 185 kDa. The existence of such an
antibody probe may facilitate the purification of the electrogenic
antiporter under denaturing conditions, in in vitro expression systems, or
in prokaryotic expression libraries.
