Arsenate Reductase II. Purine Nucleoside Phosphorylase in the Presence of Dihydrolipoic Acid Is a Route for Reduction of Arsenate to Arsenite in Mammalian Systems

Abstract

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.

Scientific Opinion on Arsenic in Food

“…Arsenate enters the cell via the phosphate carrier system and can be biotransformed enzymatically (about 50-70 % in mammals) to the more reactive arsenite (Aposhian et al, 2004) by glutathione reductase, and also by purine nucleoside phosphorylase (PNP) as proposed recently on the basis of in vitro experiments (Gregus and Nemeti, 2002;Radabaugh et al, 2002). In mammals, arsenite undergoes oxidative methylation in the liver by addition of a methyl group from S-adenosylmethionine, catalysed by arsenic-methyltransferase and resulting in the formation of methylarsonate (Figure 13).…”

Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies

“…10 Once inhaled or ingested, inorganic arsenic is transformed to organic arsenic via a series of enzyme-catalyzed successive reduction and methylation steps (Scheme 1). 11,12 Initially believed to be a detoxification mechanism, it has been realized that the organic trivalent arsenic species monomethylarsonous acid (MMA III ) and dimethylarsinous acid (DMA III ) are the most toxic of all arsenic metabolites detected in human and animal urine. [13][14][15][16][17] Variations at certain points in the human arsenic methyltransferase (AS3MT) gene may cause differences in methylating ability between individuals, 18 but data suggests a standard profile of urinary arsenic humans as 10-30% inorganic, 10-20% MMA (III+V) , and 60-80% DMA (III+V) .…”

Metabolism and toxicity of arsenic in human urothelial cells expressing rat arsenic (+3 oxidation state)-methyltransferase

“…In rats and hamsters, DMAs is further methylated to yield trimethylarsine oxide (TMAs V O) (Yamauchi and Yamamura, 1985;Yoshida et al, 1998). Two classes of enzymes are involved in the methylation of iAs, including As V -reductases (Gregus and Nemeti, 2002;Radabaugh et al, 2002;Zakharyan and Aposhian, 1999;Zakharyan et al, 2001) and As III -methyltransferases Zakharyan et al, 1995). The metabolic conversion of iAs is generally recognized to be a determinant of the toxic and carcinogenic effects of this metalloid.…”