Abstract
The affinity for K(+) of silkworm Na(+)/K(+)-ATPase, which is composed of α and β subunits, is remarkably lower than that of mammalian Na(+)/K(+)-ATPase, with a slightly higher affinity for Na(+). Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na(+) and K(+) of Na(+)/K(+)-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na(+) and K(+) of Na(+)/K(+)-ATPase. Cloned silkworm α and cloned rat β1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na(+)/K(+)-ATPase, to construct a hybrid Na(+)/K(+)-ATPase, in which the silkworm β subunit was replaced with the rat β1 subunit. The hybrid Na(+)/K(+)-ATPase increased the affinity for K(+) by 4.1-fold and for Na(+) by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm β subunit did not affect the K(+) affinity. These results support the involvement of the β subunit in the Na(+) and K(+) affinities of Na(+)/K(+)-ATPase.