The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMA(V) reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMA(V) reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMA(V) reductase activity with K(m) and V(max) values comparable to those of human liver MMA(V) reductase. The partially purified human liver MMA(V) reductase had glutathione S-transferase (GST) activity. MMA(V) reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMA(V) reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMA(V) reductase and hGSTO 1-1 are identical proteins.

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An arsenate reductase has been partially purified from human liver using ion exchange, molecular exclusion, hydroxyapatite chromatography, preparative isoelectric focusing, and electrophoresis. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, two bands were obtained. One of these bands was a 34 kDa protein. Each band was excised from the gel and sequenced by LC-MS/MS, and sequest analyses were performed against the OWL database SWISS-PROT with PIR. Mass spectra analysis matched the 34 kDa protein of interest with human purine nucleoside phosphorylase (PNP). The peptide fragments equal to 40.1% of the total protein were 100% identical to the corresponding regions of the human purine nucleoside phosphorylase. Reduction of arsenate in the purine nucleoside arsenolysis reaction required both PNP and dihydrolipoic acid (DHLP). The PNP rate of reduction of arsenate with the reducing agents GSH or ascorbic acid was negligible compared to that with the naturally occurring dithiol DHLP and synthetic dithiols such as BAL (British anti-lewisite), DMPS (2,3-dimercapto-1-propanesulfonate), or DTT (alpha-dithiothreitol). The arsenite production reaction of thymidine phosphorylase had approximately 5% of such PNP activity. Phosphorylase b was inactive. Monomethylarsonate (MMAV) was not reduced by PNP. The experimental results indicate PNP is an important route for the reduction of arsenate to arsenite in mammalian systems.