Js. Lee et al., FUNCTIONAL EXPRESSION AND SEGMENTAL LOCALIZATION OF RAT COLONIC K-ADENOSINE TRIPHOSPHATASE, The Journal of clinical investigation, 96(4), 1995, pp. 2002-2008
A putative cDNA for the colonic K-ATPase has recently been cloned (Cro
wson, M. S., and G. E. Shull, 1992, J. Biol, Chem. 267:13740-13748). C
onsiderable evidence exists that there are two K-ATPases and active K
absorptive processes in the rat distal colon: one that is ouabain sens
itive and the other ouabain insensitive. The present study used the ba
culovirus expression system to express K-ATPase activity in insect Spo
doptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), devel
oped against a fusion protein produced from the 327 nucleotide fragmen
t from 5' coding region of the putative K-ATPase cDNA, to identify the
specific localization of the K-ATPase protein. K-ATPase activity (28.
7+/-1.2 nmol inorganic phosphate/mg protein min) was expressed in plas
ma membranes isolated from Sf 9 cells infected with baculovirus contai
ning recombinant DNA with the putative K-ATPase cDNA, K-m for K for th
e K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibit
ed by ouabain (1 mM); while the K-i for vanadate inhibition was 8.3 mu
M. Western blot analysis with the M-1 antibody identified a 100-kD pr
otein in apical membranes prepared from distal, but not proximal, rat
colon. Immunohistochemical studies with M-1 antibody localized K-ATPas
e only in the apical membrane of surface cells, while an mAb (c464.6)
against Na,K-ATPase localized basolateral membranes of both surface an
d crypt cells of rat distal colon, In conclusion, the putative K-ATPas
e cDNA encodes an ouabain-insensitive K-ATPase that is present only in
the apical membrane of surface cells of rat distal colon.