Expression and characterization of a cDNA for rat kidney biliverdin reductase. Evidence suggesting the liver and kidney enzymes are the same transcript product.
Biliverdin reductase is a unique dual cofactor- and pH-dependent enzyme that converts biliverdin to bilirubin and displays extensive inter-organ pI and molecular weight microheterogeneity. Presently we have explored the molecular basis for these properties. The amino acid composition and the sequences of NH2 termini plus five tryptic fragments of purified rat liver and kidney enzymes were obtained. A 62-nucleotide DNA probe was designed and in combination with antibody was used to screen a rat kidney cDNA library. A cDNA sequence of 1108 base pairs (bp) containing an 885-bp open reading frame was generated. The cloned cDNA probe detected a single mRNA of approximately 1500 bp in liver and kidney. The open reading frame encodes a 295 amino acid protein. Methionine and aspartic acid residues at positions 1 and 2 of the deduced protein are removed during processing. The deduced amino acid composition of the mature protein closely matched that of the purified rat liver and kidney enzymes. All liver peptides were found in the deduced amino acid sequence of kidney enzyme and the NH2 termini of both enzymes were identical. The expressed protein co-migrated with purified reductase and was recognized by antiserum to the enzyme. The expressed reductase displayed two distinct pH optima using a different cofactor at each pH: NADH at the lower pH 6.7-6.9 range and NADPH at pH 8.5-8.7. The findings suggest that the liver and kidney enzymes are the products of the same transcript(s) and that their microheterogeneity may reflect tissue-specific post-translational modifications.
Fakhrai
H
H
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Maines
M D
MD
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D014158:Transcription, Genetic
D000596:Amino Acids; D012333:RNA, Messenger; D009243:NAD; D011061:Poly A; D009249:NADP; D012313:RNA; D004247:DNA; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase