Expression of Na+/K+-ATPase and H+/K+-ATPase fusion proteins in Sf-9 insect cells
The baculovirus-directed expression system has been utilized extensively to express a variety of soluble and transmembrane polypeptides in infected Sf-9 insect cells. In most instances the expressed polypeptides receive the proper post-translational modifications and assume a functionally active conformation even when multimerization is required. Recently, reconstitution of ouabain-inhibitable Na+/K+-ATPase activity has been demonstrated in Sf-9 insect cells coexpressing baculovirus induced otl and β1-subunit isoforms (1). Infections with recombinant baculovirus expressing the a or β-subunit alone showed no appreciable level of ouabain-sensitive Na+/K+-ATPase activity although both subunits were delivered to the plasma membrane independent of one another. Additional kinetic studies of uninfected insect cells demonstrated negligible levels of endogenous Na+/K+-ATPase activity and an absence of SCH 28080-inhibitable H+/K+-ATPase activity (data not shown). Thus, cultured Sf-9 insect cells represent an attractive model system for functional studies in which recombinant α-subunits can be expressed in combination with specific β-subunit isoforms and assayed for altered enzymatic activity under controlled reaction conditions. At present, the exact polypeptide regions and residues that comprise the ligand binding domains of the countertransporting P-type ATPases are unresolved. Through this current study a greater understanding of the regions of the α-subunit involved in cation sensitivity (Na+ vs. H+), inhibitor binding (ouabain vs. SCH 28080), and β-subunit interaction can be ascertained.
KeywordsATPase Activity Recombinant Baculovirus Altered Enzymatic Activity Attractive Model System Integrin Associate Protein
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