Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood.
Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4 ± 2% residual activity and CN type II patient B had 38 ± 2% residual activity.
Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 ± 0.9 μM and 7.9 ± 5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 ± 23 μM.
At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced. (J. Clin. Invest. 1994. 94:2385–2391.) Key words: hereditary diseases · site directed mutagenesis · enzyme-linked immunosorbent assay · enzyme kinetics · enzyme activity. (Reproduced from The Journal of Clinical Investigation, 1994, vol. 94, pp. 2385–2391 by copyright permission of The Society for Clinical Investigation.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.