Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit.
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)
Chow
D C
DC
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Browning
C M
CM
Forte
J G
JG
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002414:Cations, Monovalent / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004350:Drug Residues / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D011817:Rabbits; D013270:Stomach / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002414:Cations, Monovalent; D004220:Disulfides; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D003545:Cysteine