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Adams G , Tillekeratne M , Yu CL , Pestov NB , Modyanov NN
Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells
Biochemistry. 2001 May 15;40(19) :5765-5776
PMID: ISI:000168635900021   
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We previously demonstrated that the alpha -subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta -subunit (beta HK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-beta HK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-beta HK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and beta HK. 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 Arp1al1-beta HK complex were analyzed. The Atp1al1-beta HK 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 similar to 116 muM K+. The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K-i values in K+-free medium of similar to 64 muM and similar to 93 muM, 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 similar to 24 mM in the absence of added K+ and with K-i within the range of 60-70 mM in the presence of greater than or equal to1 mM K+. Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-beta HK 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.
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