The long-term objective of this laboratory's research is to provide a better understanding of the conjugation of bilirubin by the microsomal enzyme, UDP-glucuronosyltransferase (EC 2.4.1.17) and its ontogeny. In neonatal life there is a temporary deficiency in the conjugation of bilirubin, and in the Crigler-Najjar Syndromes, I and II the conjugating enzyme is absent or defective. Such clinical circumstances result in non- conjugated hyperbilirubinemia which can be either lethal or in surviving infants produce a bilirubin encephalopathy characterized by cerebral palsy, eighth nerve deafness and cognitive dysfunctioning. The animal model of impaired conjugation of bilirubin is the Gunn rat and is to be used in these investigations. The bilirubin transferase enzyme, (B-UDPGt), will be isolated to homogeneity from normal rat liver after its selective induction by clofibrate administration. The """"""""activator"""""""" peptide of the B-UPDGt will be isolated from the solubilized liver microsomes from the KCI gradient elutions of the initial anion-exchange chromatography of the B-UPDGt. These activator fractions after pooling and concentration are to be further purified by molecular sieving chromatography, and fast protein liquid chromatography on a Mono Q exchange column with a 0.1 to 0.5 M KCI gradient. The eluted activator is then isolated by preparative polyacrylamide gel electrophoresis and electroeluted from the gel. By such isolation technology sufficient material will be available to raise antibodies to the purified activator for immunocytochemical surveillance of its tissue distribution in other organs, and its ontogeny can be documented in fetal and neonatal animals. An ELISA assay is to be developed for quantization of the activator that will permit studies of its regulation by exogenous inducers and endogenous hormonal control. Parallel studies of the purified activator peptide will include its amino acid analysis, and peptide sequencing. The partial amino acid sequencing will be followed by synthesis of base-paired oligonucleotides to be used for library screening, with cDNA cloning and ultimately predicted amino acid composition from a full-coding length cDNA. Human liver samples are to be similarly studied from """"""""residual tissue"""""""" and potential biopsies so that the presence or absence of the activator peptide can be determined particularly in patients with Gilbert's syndrome in addition to the Crigler-Najjar disorders. The alternative pathway for bilirubin conjugation and excretion that utilizes cytochrome P-450 glutathione transferase is to be delineated in the B-UDPGt deficient Gunn rat. The glutathione conjugates of bilirubin in bile have been identified and characterized. Induction of this pathway by non-carcinogenic agents is to be studied for it may provide an effective treatment of life-threatening hyperbilirubinemias of man.
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