Expression and characterization of a cDNA for rat kidney biliverdin reductase. Evidence suggesting the liver and kidney enzymes are the same transcript product.

H Fakhrai,M.D Maines

doi:10.1016/s0021-9258(19)50627-5

Abstract

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.

Highlights

Biliverdin reductase is a unique dual cofactor- and ized for catalytic properties, biliverdin reductase remains pH-dependent enzyme that converts biliverdin tobili- unique in having two distinct pHoptima, 6.7-6.9 and 8.7, and rubin anddisplays extensive inter-organPIand molec- using a different cofactor at each pH, NADH is used in the ular weight microheterogeneity
The enzyme, which was first purified in a stable amino acid composition and thesequences of NHz ter- homogenous form from rat liver
Tohpeen reading frameencodes erty which was attributed to theenzyme having three reactive cysteine groups [7].The cysteine residues were implicated ( 7, 8) to be directly involved in enzymatic activity based on the observation that pyridine nucleotide coenzymes plus substrate provided full protection against potent inhibition of activity by thiol reagents

Summary

Introduction

Biliverdin reductase is a unique dual cofactor- and ized for catalytic properties, biliverdin reductase remains pH-dependent enzyme that converts biliverdin tobili- unique in having two distinct pHoptima, 6.7-6.9 and 8.7, and rubin anddisplays extensive inter-organPIand molec- using a different cofactor at each pH, NADH is used in the ular weight microheterogeneity. Prior to this study information was not available on the primary structure of the protein and the location of the 3 cysteine residues, which might contribute tothe unprecedented pH-dependent cofactor requirement of the enzyme. It was not understood whether inter-organ heterogeneity of the enzyme reflects expression of multiple transcripts. In mammals andcertain species of fish, biliverdin, the product of heme degradation by heme oxygenase isozymes [14], is converted to bilirubin This reaction is catalyzed by the the enzyme and toexplore the molecular basis of inter-organ biliverdin reductase heterogeneity. Our data suggest that the liver and kidney enzymes are similar transcript products

Methods

Results

Conclusion