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.
Site-specific mutagenesis was employed to investigate the proposed ~nt~bution of proton-donating residues (Glu, Asp) in the membrane domains of bovine rhodopsin to protonation of the Schiff base-linking protein and chromophore or to wavelength modulation of this visual pigment. Three point-mutations were introduced to replace the highly conserved residues Asp,, by Asn (D&I), Glu,,, by Gln &Q) or Glu,, by Asp (E,,D), respectively. All 3 substitutions had only marginal effects on the spectral properties of the final pigment (<3 nm blue-shift relative to native rhodopsin). Hence, none of these residues by itself is specifically involved in Schiff base protonation or wavelength modulation of bovine rhodopsin.
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