Biliverdin reductase (BVR) is essential to life in the placental animals whereby biliverdin, the immediate product of heme oxygenase activity, is converted to bilirubin and excreted through the maternal circulation. In comparison to other enzymes of heme metabolism little is known about BVR, and it was only in 1981 that our laboratory succeeded in purifying and characterizing the enzyme from rat liver. BVR is unique among all eukaryotic enzymes characterized to date in having 2 pH optima (6.7-7.0 and 8.5-8.7), using a different cofactor at each pH. The enzyme is -SH-dependent and is inhibited in vivo by toxic chemicals such as bromobenzene, mercury and cis-platinum, as well as biliverdin. More recently we reported on the multiplicity of the enzyme. In the rat kidney, liver and the spleen, up to 10 variants of the enzyme were detected. The variants could be grouped in 3 molecular weights (Mr=30,400, 30,700, 31,400) and 5 major isoelectric points (pI=6.23, 5.91, 5.76, 5.61, 5.48). Our initial studies suggest that variants differ in catalytic activity, amino acid composition and response to the environmental toxin. The variant with Mr=30,400, pI=6.23 was severely depleted in the liver and kidneys of bromobenzene-treated rats (24 h). This form was not present in the spleen and the BVR profile in this organ was not affected by bromobenzene treatment. The overall objective of the proposed research is to further characterize the variants at the molecular, biochemical and toxicological levels. Two variants of the rat kidney BVR, Mr=30,400, pI=6.23 and Mr=30,700, pI=5.48, have been selected as the focus of the proposed studies.
The specific aims are: 1) To characterize the selected BVR variants for their kinetic and enzymatic properties. 2) To characterize, by chemical modification approach, the active site of BVR variants. 3) To determine the molecular basis for enzyme heterogeneity, including the investigation of whether the variants represent products of multiple transcripts or different post-translational modifications. 4) To isolate and sequence genomic clone(s) encoding BVR and determine whether the variants represented by multiple transcripts are derived from different genes or alternative processing of the mRNA from a single gene. 5) To elucidate at the molecular level the mechanism of change in expression of the variants caused by in vivo exposure to bromobenzene and possibly by other toxic agents. 6) To examine whether the phenomenon of multiplicity extends to human tissues and other rat organs (brain and testes).
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