Purine Nucleoside Phosphorylase as a Cytosolic Arsenate Reductase

• Published in: Toxicological sciences Vol. 70; no. 1; pp. 13 - 19
• Main Authors: Gregus, Zoltán, Németi, Balázs
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Cary, NC Oxford University Press 01.11.2002
• Subjects: Animals; arsenate; Arsenates - metabolism; arsenite; Arsenite Transporting ATPases; Arsenites - metabolism; Biological and medical sciences; Catalysis; Cattle; Chemical and industrial products toxicology. Toxic occupational diseases; Cricetinae; Cytosol - enzymology; Enzyme Inhibitors - pharmacology; Guinea Pigs; In Vitro Techniques; Ion Pumps - metabolism; Liver - enzymology; Male; Medical sciences; Mesocricetus; Metals and various inorganic compounds; Mice; Mice, Inbred Strains; Multienzyme Complexes - metabolism; Nucleosides - pharmacology; purine nucleoside phosphorylase; Purine-Nucleoside Phosphorylase - antagonists & inhibitors; Purine-Nucleoside Phosphorylase - metabolism; Rabbits; Rats; Rats, Wistar; reduction; Species Specificity; Spleen - enzymology; Sulfhydryl Reagents - pharmacology; thiols; Toxicology; Purine nucleoside; Rat; Liver; Guanosine; Glycosyltransferases; Rabbit; Cytosol; Mechanism; Chemical reduction; Guinea pig; Arsenic V Compounds; Thiol; Arsenates; Enzyme; Arsenic III Compounds; Transferases; Interspecific comparison; Rodentia; Detoxification; Lagomorpha; Metabolism; Purine-nucleoside phosphorylase; Arsenites; Vertebrata; Mammalia; Mouse; Medium effect; Animal; Inosine; Ribonucleoside; Pentosyltransferases; Hamster
• DOI: 10.1093/toxsci/70.1.13
• ISSN: 1096-6080; 1096-0929

The findings of the accompanying paper (Németi and Gregus, Toxicol. Sci. 70, 4–12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate ― guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation–atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 μM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.