Functional expression of gastric H,K‐ATPase using the baculovirus expression system

Abstract

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.

The β-Subunits of Na+,K+-ATPase and Gastric H+,K+-ATPase Have a High Preference for Their Own α-Subunit and Affect the K+ Affinity of These Enzymes

“…The molecular mass of all ␣-subunits expressed was similar to that of the isolated ␣-subunits. However, the recombinant ␤-subunits were less glycosylated than the isolated ␤-subunits, as has been reported before (9,52). Expression levels of the subunits of each hybrid ATPase were comparable to those of Na ϩ ,K ϩ -ATPase and H ϩ ,K ϩ -ATPase, respectively.…”

“…All ␣-subunits were cloned downstream of the polyhedrin promoter, and all ␤-subunits downstream of the p10 promoter. As mock, the baculovirus DZ1, only expressing ␤-galactosidase, was used (9).…”

“…Preparation of Membranes-Sf9 cells were grown at 27°C in 100-ml spinner flask cultures (9). For production of the ATPases subunits 1.0 -1.5 ϫ 10 6 cells/ml were infected at a multiplicity of infection of 1-3 in Xpress medium (BioWittaker, Walkersville, MD) containing 1% ethanol (37) and incubated for 3 days.…”

“…This formation of a complex between ␣-and ␤-subunits is also essential for enzyme activity (7)(8)(9) and occurs before the subunits are transported from the endoplasmic reticulum to the plasma membrane (10). Lemas et al (11) showed that the carboxylterminal 161 amino acids of the Na ϩ ,K ϩ -ATPase ␣-subunit are sufficient for assembly with the ␤-subunit.…”

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…Sf 9 cells were grown at 27°C in 100-ml spinner flask cultures as described by Klaassen et al [16]. For expression of the ATPases, 1.5.10 6 cells.ml −1 were infected with the proper virus at a multiplicity of infection of 1-3 in the presence of 1% (v/v) ethanol and 0.1% (w/v) pluronic F-68 (ICN, Aurora, OH, USA) in Xpress medium (Biowittaker, Walkersville, MD, USA) as described before [17].…”