Bromobenzene-mediated alteration in activity and electrophoretic pattern of biliverdin reductase variants in rat...

Here we report on the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat kidney and describe selective changes in the tissue profile of the variants after bromobenzene treatment (2 mmol/kg, subcutaneously, 24 hr). Using two-dimensional electrophoresis and isoelectric focusing, two major molecular mass species, Mr 30,400 and 30,700, a minor form of Mr 31,400, and five net-charge groups of pI = 6.23, 5.91, 5.77, 5.61, and 5.48 were detected; the net-charge variants with pI = 5.61 and 5.77 were the most abundant forms. The Mr 30,400 form was the main component of two isoelectric focusing bands with pI = 6.23 and 5.91, and the relative amounts of these net-charge variants was severely decreased in the kidneys of bromobenzene-treated rats. The effect of bromobenzene in vivo could not be duplicated by in vitro experiments involving the direct treatment of purified enzyme with bromobenzene, or incubation of the purified preparation with bromobenzene in the presence of a NADPH-dependent microsomal drug-metabolizing system. Bromobenzene treatment did not alter the immunochemical properties of biliverdin reductase variants, as judged by the similarity of isoelectric focusing patterns of preparations on a Western blot using antibody raised against a rat liver total biliverdin reductase preparation. The treatment, however, caused an alteration in the kinetic properties of the enzyme, and the activity with NADH appeared to be selectively decreased. The possible mechanisms involved in the expression of multiple forms of the reductase and the biological significance of the multiplicity, as well as the change in composition caused by bromobenzene, are discussed.