Multiple forms of biliverdin reductase: modification of the pattern of expression in rat liver by bromobenzene.
Rat liver biliverdin reductase was purified from control and bromobenzene-treated rats and was designated as C-BVR-T and B-BVR-T, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the existence of two molecular weight variants (30,100 and 29,800) in C-BVR-T but only one form (30,100) in B-BVR-T. Western immunoblotting confirmed that both molecular weight variants were biliverdin reductase. Nondenaturing electrophoresis separated C-BVR-T and B-BVR-T preparations into groups of four variants, designated as BVR ND1 to ND4. However, the C-BVR-T preparation contained three major forms (BVR ND1, ND2, and ND3) while the B-BVR-T preparation contained two major forms (BVR ND2 and ND3). In vitro treatment of biliverdin reductase preparations with either bromobenzene or dithiothreitol did not interconvert the variants of the enzyme. QAE-Sepharose anion-exchange chromatography was used to isolate the ND2 and ND3 variants for physiochemical analysis. The amino acid composition of the variants was rather similar except for their Tyr content. Also, the peptide maps were similar except for a series of moderately early chromatographic peaks. These findings implied secondary modifications to the protein rather than substantial differences in primary structure. The pH-dependent cofactor requirements for enzyme activity were examined. Both variants exhibited 2 pH optima that were cofactor dependent; maximum activity with NADPH and NADH was observed at pH 8.5 and 6.7, respectively. However, both variants exhibited a higher catalytic rate with NADH than with NADPH at their pH optima. Furthermore, BVR ND3 exhibited a higher catalytic rate than BVR ND2 with either cofactor throughout the pH range 6.5-9.
Huang
T J
TJ
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Trakshel
G M
GM
Maines
M D
MD
D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D015153:Blotting, Western; D001969:Bromobenzenes / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats
D000596:Amino Acids; D001969:Bromobenzenes; D007527:Isoenzymes; C032036:bromobenzene; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase