BIOCHEMICAL-CHARACTERIZATION OF THE HUMAN ARSENITE-STIMULATED ATPASE (HASNA-I)

Arsenic is a potent toxin and carcinogen. In pro karyotes, arsenic det oxification is accomplished by chromosomal and plasmid-borne operon-en coded efflux systems. We have previously reported the cloning of hASNA -I, a human homologue of arsA encoding the ATPase component of the Esc herichia 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 abs ence 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; anti monite 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 pro duced a single monomeric 37-kDa band on SDS-polyacrylamide gel electro phoresis (PAGE) and two distinct species when analyzed using nondenatu ring PAGE. Second, chemical cross-linking of the 63-kDa GST-hASNA-I re sulted in the formation of dimeric and tetrameric protein forms, The r esults indicate that hASNA-I is a distinct human arsenite-stimulated A TPase belonging to the same superfamily of ATPases represented by the E. coli ArsA protein.