Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells.
We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.
Adams
G
G
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
Tillekeratne
M
M
Yu
C
C
Pestov
N B
NB
Modyanov
N N
NN
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001667:Binding, Competitive; D002384:Catalysis; D002460:Cell Line / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007093:Imidazoles / Q000494:pharmacology; D046911:Macromolecular Substances; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000502:physiology; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis; D012964:Sodium / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*
D004791:Enzyme Inhibitors; D007093:Imidazoles; D046911:Macromolecular Substances; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins; C035235:Sch 28080; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; C088641:ATP12A protein, human; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium