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.
Kurdi-Haidar
B
B
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
Heath
D
D
Aebi
S
S
Howell
S B
SB
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