Enzymic Methylation of Arsenic Compounds: Assay, Partial Purification, and Properties of Arsenite Methyltransferase and Monomethylarsonic Acid Methyltransferase of Rabbit Liver

Abstract

A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate (MMA) by rabbit liver methyltransferases. The assay has been validated by separating, identifying, and measuring the products of the reaction using chloroform extraction, ion exchange chromatography, TLC, or HPLC. The enzymes involved in this pathway, arsenite methyltransferase and MMA methyltransferase, have been purified approximately 2000-fold from rabbit liver. After gel electrophoresis, a single band is obtained with both enzyme activities in it. The pH optima for purified arsenite methyltransferase and monomethylarsonic acid methyltransferase are 8.2 and 8.0, respectively. A thiol, S-adenosylmethionine, and arsenite are required for the partially purified arsenite methyltransferase that catalyzes the synthesis of monomethylarsonate. A different enzyme activity that catalyzes the methylation of monomethylarsonate to dimethylarsinate also requires SAM and a thiol. Even though arsenite methyltransferase and monomethylarsonate methyltransferase have different substrates, pH optima, and saturation concentrations for their substrates, whether the two activities are present on one protein molecule or different protein molecules is still uncertain. Both activities have a molecular mass of 60 kDa as determined by gel exclusion chromatography. There is no evidence at the present time for these enzyme activities being on different protein molecules. Neither arsenate, selenate, selenite, or selenide are methylated by the purified enzyme preparations. Results from the use of crude extracts, often called cytosol, to study the properties of these methyltransferases dealing with arsenic species should be viewed with caution since such crude extracts contain inhibiting and other interfering activities.

A Novel S-Adenosyl-l-methionine:Arsenic(III) Methyltransferase from Rat Liver Cytosol

“…These results are consistent with evidence that trivalent arsenicals are the preferred substrate for this enzyme and that the pentavalent arsenicals must be reduced enzymatically or nonenzymatically before methylation (7,11,41). Based on estimated molecular weights, the protein purified from rat liver cytosol (ϳ42.000) is probably not identical with the enzyme purified from rabbit liver cytosol (ϳ60,000) that catalyzes the methylation of arsenite and methylarsonous acid (10,11). S-Adenosyl-Lmethionine:arsenic(III) methyltransferase from rat liver differs kinetically from arsenic methyltransferases purified from rabbit or hamster liver cytosol.…”

“…A critical factor in the success of this purification scheme was inclusion of GSH and DTT in all buffers. Earlier work demonstrated that GSH was critical to the maintenance of arsenic methylating activity in an in vitro reaction system that contained rat liver cytosol (33,34) and that DTT promoted the dimethylation reaction catalyzed by the purified rabbit liver protein (10). S-Adenosyl-L-homocysteineSepharose affinity chromatography yielded a single Coomassie Blue staining band on SDS-PAGE (Fig.…”

“…The high yield of activity in the purification scheme may be due to removal of endogenous inhibitors or alteration of the enzyme: DTT ratio. Others have noted the presence of endogenous inhibitors for arsenite and methylarsonous acid methyltransferases in cytosol prepared from the livers of rabbits or marmosets (10,40). The pattern of appearance of the methylated arsenicals suggests that the initial reaction forms methylated arsenic from arsenite; a second reaction then forms dimethylated arsenic from methylated arsenic.…”

“…Cullen and co-workers (4) summarized the conversion of inorganic arsenic into these methylated products in a reaction scheme which incorporates oxidative methylation and the cycling of arsenic between the pentavalent (As V ) 1 Because reduction of arsenic to trivalency is a prerequisite for its oxidative methylation, pentavalent arsenicals are reduced by endogenous thiols such as glutathione (GSH) (5,6) or by As V reductases (7)(8)(9). A protein has been purified from rabbit liver cytosol that catalyzes the methylation of both arsenite and methylarsonous acid (10,11); however, this protein has not been sequenced. These activities are designated arsenite methyltransferase (EC 2.1.1.137) and methylarsonite methyltransferase (EC 2.1.1.138), respectively.…”

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.…”