title: Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells.
abstract: It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione-conjugated arsenicals can still react with some proteins in hepatic cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Mizumura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Watanabe
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Seishiro
authors.initials: S
authors.lastname: Hirano
mesh_terms: D000595:Amino Acid Sequence; D001151:Arsenic / Q000378:metabolism*; D018053:Arsenites / Q000378:metabolism*; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018053:Arsenites; D009363:Neoplasm Proteins; D005978:Glutathione; C015001:arsenite; D001151:Arsenic