Biochemical characterization of the human arsenite-stimulated ATPase (hASNA-I).
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 Vmax; 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.
Department of Medicine and the University of California, San Diego Cancer Center, University of California, San Diego, La Jolla, California 92093-0058, USA. bhaidar@ucsd.edu
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Kurdi-Haidar
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Heath
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Howell
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D018053:Arsenites / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D018053:Arsenites; D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; C015001:arsenite