Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.27309 |
| Date | January 1996 |
| Creators | Dupuis, Lucie. |
| Contributors | Gravel, Roy A. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001566638, proquestno: MQ29684, Theses scanned by UMI/ProQuest. |
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