Abstract
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 K(m) 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, K(m) = 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.
Original language | English |
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Pages (from-to) | 2385-2391 |
Number of pages | 7 |
Journal | Journal of clinical investigation |
Volume | 94 |
Issue number | 6 |
DOIs | |
Publication status | Published - Dec 1994 |
Keywords
- enzyme activity
- enzyme kinetics
- enzyme-linked immunosorbent assay
- hereditary diseases
- site directed mutagenesis