AJP - Cell Physiology, Vol 263, Issue 1 C39-C46, Copyright © 1992 by American Physiological Society
Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit
D. C. Chow, C. M. Browning and J. G. Forte
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was
irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and
dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase,
high concentrations of reagents, at moderately elevated temperatures, were
required to inactivate H(+)-K(+)-ATPase, suggesting relative
inaccessibility of the responsible disulfide bonds. Resistance against
inactivation was conferred by monovalent cation activators of
K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of
K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater
than K+ greater than Rb+) and concentration to their respective affinities
for stimulating enzymatic activity, suggesting that the K(+)-bound form of
the enzyme is more resistant to reduction than the free enzyme.
Furthermore, Na+ antagonized the protective effect of K+. Labeling studies
using fluorescein-maleimide indicated that 60-70% of the cysteine residues
in the beta-subunit are in the oxidized form. Coupled with primary sequence
data, this suggests that three disulfide bonds are present in the native
beta-subunit. In contrast, less than 10% of the cysteine residues in the
alpha-subunit are in the oxidized form. Kinetic studies showed that the
2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated
with a reduction of disulfide groups in the beta-subunit, while there was
no significant change in the alpha-subunit. We conclude that reduction of
disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of
K+ to the enzyme confers a resistance to disulfide bond reduction, and the
responsible disulfide bonds are present in the beta-subunit.(ABSTRACT
TRUNCATED AT 250 WORDS)
