title: Molecular differences between rat-liver and rat-kidney biliverdin reductase. Implications for their in vivo regulation.
abstract: Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme.
authors.affiliation: Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
authors.forename: O
authors.initials: O
authors.lastname: Cascone
authors.forename: R B
authors.initials: RB
authors.lastname: Frydman
authors.forename: P
authors.initials: P
authors.lastname: Ferrara
authors.forename: M L
authors.initials: ML
authors.lastname: Tomaro
authors.forename: J
authors.initials: J
authors.lastname: Rosenfeld
mesh_terms: D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis* / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D014357:Trypsin; D003488:Cyanogen Bromide