Functional Analysis Of Na~+-transporting Genes SmrL And Nhad1

smrh is a Na+/H+antiporter gene cloned from total DNA of enrichment culture. In this study,5amino acid residues associated with transmembrane segments of SmrL were screened for their possible role in the ion translocation and pH regulation. Substitutions L6A、E13A、 Y59A completely not grow in the LBK medium containing0.25M NaCl. Substitutions L6A, E13A, Y59A, E13D, K21A can’t grow in the LBK medium containing0.01M LiCl. L6A, E13D grow better in basic medium of pH7than the wild type. Na+/H+antiporter activity of all mutants reduced except for E13D, and L6A,Y59A results in an alkali shift of its pH profile, while K21A and T63A results in an acidic shift of0.5units. As to the activity of Li+/H+.antiporter, T63A completely abolished, L6A, E13A, Y59A, E13D, K21A reduced, L6A and E13A results in an alkali shift of its pH profile, while K21A and Y59A results in an acidic shift of0.5units.Two genes nhadl and nhar encoding Na+/H+antiporter were subcloned from the recombinant plasmid of E.coli KNabc/pUCNHD, and were transformed into E.coli KNabc. The result showed that KNabc/pUCnhad1including the nhad1gene could grow in the LBK medium containing0.2M NaCl, but KNabc/pUCnhar ncluding the nhar gene can’t grow in the same condition. The nhadl enable KNabc/pUCnhadl grow in the LBK medium containing up to0.5M NaCl or0.2M LiCl. Everted membrane vesicles prepared from KNabc/nhadl exhibited high Na+/H+as well as Li+/H+antiporter activity, which was pH-dependent with the highest activity at pH7.0, and no K+/H+antiporter activity was exhibited. The Na+/H+antiporter activity of KNabc/Nhadl is nine times than that of KNabc/pUCNHD,and the Km value of Nhad1for Na+and Li+is1.45and2.94, respectively.