Abstract
The 76-kDa NtpI subunit constitutes the membrane-embedded V(0) moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 +/- 0.03 and 0.40 +/- 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.
Highlights
Introduction of the HistidineTag into the Carboxyl Terminus of NtpI—PCR was performed with pHEexI as the template, using primer 1 (5Ј-GCTTGGCAATGGATCCTGCTTGGTATCATTCTG-3Ј) and primer 2 (5Ј-GCCGGAATTCCTAATGATGATGATGATGATGTTTTTTCTTATGATTGATGTTGACG-3Ј), resulting in the addition of six histidine residues to the carboxyl terminus of the ntpI gene product
The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase
We have identified a variant of V-ATPase in Enterococcus hirae, which characteristically transports Naϩ or Liϩ [4]
Summary
Introduction of the HistidineTag into the Carboxyl Terminus of NtpI—PCR was performed with pHEexI as the template, using primer 1 (5Ј-GCTTGGCAATGGATCCTGCTTGGTATCATTCTG-3Ј) and primer 2 (5Ј-GCCGGAATTCCTAATGATGATGATGATGATGTTTTTTCTTATGATTGATGTTGACG-3Ј), resulting in the addition of six histidine residues to the carboxyl terminus of the ntpI gene product (underlined). The negative cooperativity for the Na؉ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 ؎ 0.03 and 0.40 ؎ 0.05, respectively. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.
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