An analysis of the topography of molecular forms 1 and 3 of rat liver biliverdin reductase using polyclonal antibodies.
Rat liver biliverdin reductase exists in two molecular forms. The major one (molecular form 1) is transformed, under conditions of oxidative stress into another molecular form (molecular form 3) which is an S-S bridged dimer of form 1. The chemical modifications of the thiol, arginine and lysine residues of molecular form 1 which resulted in an inhibition of its catalytic activity did not affect the activity of molecular form 3. Rabbit polyclonal antibodies raised against form 1 did not recognize form 3. This lack of recognition persisted even when the dimer (form 3) was denatured with SDS or urea under non-reductive conditions. Reduction of form 3 with reduced thioredoxin gave the monomeric form 1, which was fully recognized by the antibodies. The latter recognized the biliverdin reductases from rat spleen and kidney to the same extent as they did with form 1. Molecular form 1 was completely inhibited by the addition of the antibodies. This inhibition was prevented by preincubation of the enzyme with either the substrate (biliverdin) or the cosubstrate (NADPH). Preincubation with the latter or with NADP+ (but not with bilirubin) strongly impaired the recognition of form 1 by the antibodies. Modification of the lysine or arginine residues of form 1 which were involved in substrate binding, impaired the interaction of the enzyme with the antibodies. The antisera blocked the enzymatic conversion of form 1 to form 3, but alkylation of the thiol residue involved in this dimerization had no effect on the interaction of form 1 with the antibodies. The lack of recognition of form 3 by the antibodies suggest that the antigenic site of the former becomes buried upon dimerization.
Frydman
J
J
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Tomaro
M L
ML
Frydman
R B
RB
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D003429:Cross Reactions; D004220:Disulfides / Q000032:analysis; D000939:Epitopes / Q000032:analysis; D005260:Female; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D010088:Oxidoreductases / Q000276:immunology / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D000936:Antigen-Antibody Complex; D004220:Disulfides; D000939:Epitopes; D007527:Isoenzymes; D046911:Macromolecular Substances; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase