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Free keywords:
ATPase, Na+/K+; ATPase, H+/K+; ATPase α-subunit; ATPase β-subunit; Chimeric enzyme
Abstract:
Two sets of chimeric beta-subunits were constructed from subunits of Torpedo californica Na+/K+-ATPase and pig gastric H+/K+-ATPase. Five unique restriction sites (SnaBI, EcoRV, MunI, SphI and EcoT22I) were created at equivalent positions of the respective cDNAs and were used as joining points for the construction. One set of chimeras (HxN series) was made by exchanging the 5' portion of the Na+/K+-ATPase beta-subunit cDNA with the corresponding portion of the H+/K+-ATPase beta-subunit cDNA at the respective joining point. Complementary constructs were also prepared (NxH series). In the HxN series, the chimera joined at the SnaBI site formed a stable trypsin resistant complex with the Na+/K+-ATPase alpha-subunit, which was functional with respect to ATP hydrolysis and pump current generation, although the activities were less than those of the complex with the Na+/K+-ATPase beta-subunit. Trypsin resistance decreased for the complex of the chimera joined at the EcoRV site. In the NxH series, the chimeras joined at the SnaBI site and the EcoRV site formed rather trypsin-resistant complexes, but the expressions of the alpha-subunits were below 50% of the control. The chimeras joined at the MunI, SphI and EcoT22I site formed complexes susceptible to tryptic digestion. None of the chimeras in the NxH series were functional. These results suggest that at least two regions of the Na+/K+-ATPase beta-subunit [SnaBI site(Tyr40) to EcoRV site(Ile89) and EcoT22I site(Cys176) to C-terminus)] are involved in stable assembly with the Na+/K+-ATPase alpha-subunit and that the cytoplasmic domain [N-terminus to SnaBI site(Tyr40)] is functionally replaceable with the corresponding domain of the H+/K+-ATPase beta-subunit.
