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Arsenic is a potent toxin and carcinogen. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. We have previously reported the cloning of hASNA-I, a human homologue of arsA encoding the ATPase component of the Escherichia coli arsenite transporter. Purified glutathione S-transferase (GST)-hASNA-I fusion protein was biochemically characterized, and its properties were compared with those of ArsA. The GST-hASNA-I exhibited a basal level of ATPase activity of 18.5 ؎ 8 nmol/min/mg in the absence of arsenite. Arsenite produced a 1.6 ؎ 0.1-fold stimulation of activity (p ‫؍‬ 0.0044), which was related to an increase in V max ; antimonite did not stimulate activity. Two lines of evidence suggest that an oligomer is the most likely native form of hASNA-I. First, lysates of human embryo kidney 293 cells overproducing recombinant hASNA-I produced a single monomeric 37-kDa band on SDS-polyacrylamide gel electrophoresis (PAGE) and two distinct species when analyzed using nondenaturing PAGE. Second, chemical cross-linking of the 63-kDa GST-hASNA-I resulted in the formation of dimeric and tetrameric protein forms. The results indicate that hASNA-I is a distinct human arsenite-stimulated ATPase belonging to the same superfamily of ATPases represented by the E. coli ArsA protein.Arsenic is a toxic metalloid whose reactive trivalent and pentavalent ions can influence a number of biochemical processes. In bacteria, arsenic detoxification is mediated by specific chromosomal (1, 2) as well as plasmid-transmissible operons (3, 4) encoding efflux systems that confer low and high level resistance to arsenic, respectively. The well characterized plasmid-borne ars operon of Escherichia coli is composed of two regulatory (arsR and arsD) and three structural (arsA, arsB, and arsC) genes. An oxyanion-dependent ATPase is encoded by the arsA gene and associates with the channel-forming transmembrane protein encoded for by arsB (5). ArsC is an arsenate reductase (6). In contrast to the plasmid-borne ars operon of E. coli, both its chromosomal ars operon and the plasmid-borne operon in Gram-positive bacteria lack arsD and arsA (7,8).In mammalian cells, although evidence for the presence of an ATP-dependent arsenite efflux system has been reported (9), none of its components have been molecularly isolated. As part of our efforts to identify genes involved in drug and arsenite resistance, we focused our efforts on the isolation of the human homologue of the bacterial ATP-binding ArsA protein, a putative component of an arsenite efflux pump in human cells.

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