Abstract
The P-type ATPase, CopB, of Enterococcus hirae is required for the copper resistance displayed by this organism and thus was postulated to be a copper pump. Using 64Cu+ and 110mAg+, we here show ATP-driven copper and silver accumulation catalyzed by CopB in native inside-out membrane vesicles of E. hirae. CopB ATPase exhibited an apparent Km for Cu+ and Ag+ of 1 microM and for ATP of 10 microM. Transport was maximal at pH 6 and had an apparent Vmax of 0.07 nmol.min-1.mg-1 for both copper and silver transport. Vanadate displayed a biphasic effect on transport: maximal inhibition was observed at 40 microM vanadate for copper transport and 60 microM for silver transport, respectively. At higher vanadate concentrations, these inhibitions were reversed. The CopB ATPase of E. hirae is thus a pump for the extrusion of monovalent copper and silver ions, with copper probably being the natural substrate.
Highlights
Typical features ofP-type A'I'Pases, for which Ca2+-ATPases are paradigms, are a protein subunit of around 100 kDa, sensitivity to vanadate, and the formation of an acylphosphate intermediate in the transport reaction
Dithiothreitol, which proved essential to measure Cu + transport, was not required for silver transport; it interfered with the Ag" measurements, probably by interacting with Ag". These results show that silver[1] is a substrate for the CopB ATPase and that dithiothreitol employed in the copper transport experiments is not required by the vesicles, but strictly serves to reduce coppet'(H) to copper(l)
We here show that CopB of E. hirae is an ATP-driven pump that can expel Cu " and Ag" from the cytoplasm
Summary
Typical features ofP-type A'I'Pases, for which Ca2+-ATPases are paradigms, are a protein subunit of around 100 kDa, sensitivity to vanadate, and the formation of an acylphosphate intermediate in the transport reaction. We have recently described one such mechanism in Enterococcus hirae that is dependent on the activity of two ATPases, CopA and CopB, that are regulated in their expression by the copper concentration 0, 2).
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