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J Biol Chem, Vol. 275, Issue 6, 3873-3878, February 11, 2000
From the Department of Biochemistry and Molecular Biology, Wayne
State University School of Medicine, Detroit, Michigan 48201
ZntA, a soft metal-translocating P1-type ATPase
from Escherichia coli, confers resistance to Pb(II),
Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein,
solubilized from membranes with Triton X-100, and purified to
homogeneity. The soft metal-dependent ATP hydrolysis
activity of purified ZntA was characterized. The activity was specific
for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity
obtained when the metals were present as thiolate complexes of cysteine
or glutathione. The maximal ATPase activity of ZntA was ~3
µmol/(mg·min) obtained with the Pb(II)-thiolate complex. In the
absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the
Cd(II)-thiolate complexes stimulate activity, suggesting that a
metal-thiolate complex is the true substrate in vivo. These
results are consistent with the physiological role of ZntA as mediator
of resistance to toxic concentrations of the divalent soft
metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first
Pb(II)-dependent ATPase discovered to date.
The ATP Hydrolytic Activity of Purified ZntA, a
Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia
coli*
*
This work was supported in part by Wayne State University
start-up funds (to B. M.), by United States Public Health Service Grants GM54102 (to B. M.) and GM55425 (to B. P. R.), and
by National Research Service Award GM18973 (to C. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-0040; Fax:
313-577-2765; E-mail: bmitra@med.wayne.edu.
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