Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems.
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.
Radabaugh
Timothy R
TR
Department of Molecular and Cellular Biology, The University of Arizona, Tucson 85721-0106, USA.
Sampayo-Reyes
Adriana
A
Zakharyan
Robert A
RA
Aposhian
H Vasken
HV
D000818:Animals; D000975:Antioxidants / Q000737:chemistry*; D053501:Arsenite Transporting ATPases; D002417:Cattle; D002845:Chromatography; D004347:Drug Interactions; D004586:Electrophoresis; D006801:Humans; D016623:Ion Pumps / Q000737:chemistry / Q000302:isolation & purification*; D008099:Liver / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000302:isolation & purification*; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*
D000975:Antioxidants; D016623:Ion Pumps; D009097:Multienzyme Complexes; D008063:Thioctic Acid; C015144:dihydrolipoic acid; D011683:Purine-Nucleoside Phosphorylase; D053501:Arsenite Transporting ATPases