Gcr. Ellisdavies et al., PHOTOLABILE AMILORIDE DERIVATIVES AS CATION SITE PROBES OF THE NA,K-ATPASE, The Journal of biological chemistry, 271(17), 1996, pp. 10353-10358
Treatment of purified canine renal Na,K-ATPase with a range of photoac
tivatable amiloride derivatives results in inhibition of ATPase activi
ty prior to illumination. Inhibition by amiloride derivatives substitu
ted on a guanidium N could not be prevented by the presence of either
K or Na; however, these cations could protect the enzyme against inhib
ition by derivatives substituted on the 5-position of the pyrazine rin
g. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NE
NMBA), the presence of monovalent cations (Na, K, and Rb) protected th
e enzyme effectively against inhibition, with concentrations in the mi
llimolar range. ATP did not prevent inhibition; furthermore, native an
d NENMBA-treated enzyme exhibited normal levels of high affinity [H-3]
ADP (and hence ATP) binding. The rate of inhibition increased with inc
reasing concentrations of NENMBA. Extensive washing of NENMBA-inhibite
d enzyme did not restore ATPase activity, showing that NENMBA has an e
xtremely slow off-rate for dissociation from its inhibitory site. Part
ially inhibited enzyme could be rapidly pelleted and resuspended in NE
NMBA-free buffer and inhibition was observed to continue, albeit at a
somewhat diminished rate, suggesting that NENMBA gains access to its i
nhibitory site after partitioning into the lipid phase rather than dir
ectly from the aqueous solution. Photolysis of NENMBA inhibited enzyme
resulted in covalent incorporation of the reagent into the ar-subunit
of the Na,K-ATPase, as observed by separation of labeled protein on a
Laemmli gel and Western analysis using a polyclonal amiloride antibod
y. Almost all of the covalent labeling could be prevented by the prese
nce of Rb in the incubation and labeling medium. These results suggest
that NENMBA inhibits the Na,K-ATPase by disruption of the cation tran
sport domain rather than the catalytic domain of the enzyme and that i
t promises to be a useful tool for cation site localization.