BINDING OF ARSENICALS TO PROTEINS IN AN IN-VITRO METHYLATION SYSTEM

The dynamics of interactions between rat liver cytosolic proteins and arsenicals were examined in an in vitro methylation system that contai ned cytosol, glutathione, S-adenosylmethionine, and 1 mu M [As-73]arse nite. After incubation at 37 degrees C for up to 90 min, low-molecular -weight components of the assay system (<10 kDa) were removed by ultra filtration and cytosolic proteins were separated by size-exclusion chr omatography on Sephacryl S-300 gel. Five As-73-labeled protein peaks w ere found in chromatographic profiles. The estimated molecular masses of As-73-labeled proteins eluting in the three earliest peaks were as follows: V-o, greater than or equal to 1000 kDa; A, 135 kDa; and B, 38 kDa. Peak C eluted immediately before the total volume (V-T) of the c hromatographic column; peak D eluted after the V-T. As-73 bound to pro teins was released by CuCl treatment and speciated by thin-layer chrom atography. Amounts and ratios of inorganic As, methyl As, and dimethyl As associated with cytosolic proteins depended upon the incubation in terval. Inorganic As was present in all protein peaks. Methyl As was p rimarily associated with peaks A and C; dimethyl As was associated wit h peaks B and C. To examine the effect of valence on the binding of me thylarsenicals to cytosolic proteins, trivalent or pentavalent C-14-la beled methyl As or dimethyl As was incubated in an in vitro system des igned to minimize the enzymatically catalyzed production of methylated arsenicals. Proteins in peaks A, B, and C bound preferentially trival ent methyl and dimethyl As. Peak D bound either trivalent or pentavale nt methyl and dimethyl As. Protein-bound inorganic and methyl As were substrates for the production of dimethyl As in an in vitro methylatio n system, suggesting a role for protein-bound arsenicals in the biomet hylation of this metalloid. (C) 1997 Academic Press.