ASSEMBLY OF THE CHIMERIC NA+ K+-ATPASE AND H+/K+-ATPASE BETA-SUBUNIT WITH THE NA+/K+-ATPASE ALPHA-SUBUNIT/

Two sets of chimeric beta-subunits were constructed from subunits of T orpedo californica Na+/K+-ATPase and pig gastric H+/K+-ATPase. Five un ique restriction sites (SnaBI, EcoRV, MunI, SphI and EcoT22I) were cre ated 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-subuni t cDNA with the corresponding portion of the H+/K+-ATPase beta-subunit cDNA at the respective joining point. Complementary constructs were a lso prepared (NxH series). In the HxN series, the chimera joined at th e SnaBI site formed a stable trypsin resistant complex with the Na+/K-ATPase alpha-subunit, which was functional with respect to ATP hydrol ysis and pump current generation, although the activities were less th an those of the complex with the Na+/K+-ATPase beta-subunit. Trypsin r esistance decreased for the complex of the chimera joined at the EcoRV site. In the NxH series, the chimeras joined at the SnaBI site and th e EcoRV site formed rather trypsin-resistant complexes, but the expres sions of the alpha-subunits were below 50% of the control. The chimera s joined at the MurtI, SphI and EcoT22I site formed complexes suscepti ble 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 Ec oT22I site(Cys176) to C-terminus)] are involved in stable assembly wit h the Na+/K+-ATpase alpha-subunit and that the cytoplasmic domain [N-t erminus to SnaBI site(Tyr40)] is functionally replaceable with the cor responding domain of the H+/K+-ATPase beta-subunit. (C) 1997 Elsevier Science B.V.