Catalytic Function of Nongastric H,K-ATPase Expressed in Sf-21 Insect Cells

Abstract

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.

The Non-gastric H,K-ATPase Is Oligomycin-sensitive and Can Function as an H+,NH4+-ATPase

“…One has to realize, however, that the circumstances of the dephosphorylation experiments (time and temperature) are so different from those of the ATPase measurements that their results can only be used in a semiquantitative way. In contrast to the present study, Na ϩ did not increase the activity of human non-gastric H,K-ATPase when co-expressed with the ␤-subunit of gastric H,K-ATPase in Sf21 cells, whereas it inhibited the K ϩ -stimulated enzyme (9). The maximal ATPase activity reported by Adams et al (9) was only 5-10% of that measured in the present study.…”

“…In contrast to the present study, Na ϩ did not increase the activity of human non-gastric H,K-ATPase when co-expressed with the ␤-subunit of gastric H,K-ATPase in Sf21 cells, whereas it inhibited the K ϩ -stimulated enzyme (9). The maximal ATPase activity reported by Adams et al (9) was only 5-10% of that measured in the present study. Adams et al (9), however, included 6 M oligomycin in their assay media, whereas we show here that this drug inhibits rat non-gastric H,K-ATPase with an IC 50 of 3 M. It is not clear whether the different results are because , or NaCl (Na ϩ ) (C) concentrations, and the incubation was continued for 3 s. The residual phosphorylation level corrected for that of mock-infected cell membranes was expressed as a percentage of the level at t ϭ 0. of species differences, a different ␤-subunit, and/or methodical differences.…”

“…More recently, this enzyme has also been studied in several expression systems including Xenopus laevis oocytes (6, 24 -27), HEK 293 cells (14,28), and the baculovirus expression system (9,29). The results of these studies are, however, rather confusing, in particular regarding the sensitivity toward ouabain and SCH 28080.…”

Identification of the β-subunit for nongastric H-K-ATPase in rat anterior prostate

“…However, detailed comparison of the capability of each of the known X-K-ATPase ␤-subunits to form a functionally active ATPase complex with the ␣ ng on coexpression in Xenopus oocytes revealed that ␤ g and ␤ 2 -like Bufo bladder ␤ are able to associate with ␣ ng much more efficiently than its real counterpart, ␤ 1 (21, 26). Formation of the active ATPase complex of ␣ ng with ␤ g , but not with ␤ 1 or ␤ 3 , was observed in baculovirus expression system (1,2). It is logical to assume that the phenomenon of preferential association of the ␣ ng with ␤ g and Bufo bladder ␤ in heterologous expression systems is based on intrinsic structural features of these particular ␤-subunits, which are designed by nature to resist the harsh environmental conditions in mammalian stomach or in frog urinary bladder and, therefore, exhibit a greater ability to survive in heterologous expression systems.…”

“…For rat distal colon and kidney, it was reported to be either ␤ 1 (14,34) or, in sharp contrast, ␤ 3 (52). On the other hand, in heterologous expression systems, the functional expression of nongastric H-K-ATPase can be supported by various X-K-ATPase ␤-subunit isoforms (1,2,6,15,18,21,24,27,30,31,40).…”