Abstract
A simple, rapid assay was developed to diagnose holocarboxylase synthetase deficiency. Holocarboxylase synthetase first catalyzes the formation of biotinyl-AMP from biotin and ATP, an activity designated as biotinyl-AMP synthetase. In the second step of the reaction, biotin is transferred from biotinyl-AMP to the enzymatically inactive apocarboxylase to form an active holocarboxylase. The assay for holocarboxylase synthetase activity therefore requires a protein apocarboxylase substrate which is not readily available. In the assay for biotinyl-AMP synthetase, hydroxylamine reacts nonenzymatically with the product of the enzymatic reaction, biotinyl- AMP, to form biotinylhydroxamate. At the end of the reaction, unreacted radioactive biotin substrate, which is negatively charged at neutral pH, is bound to an anion-exchange resin and a neutral radioactive biotinylhydroxamate product in the supernatant is counted. In fibroblasts from 11 patients with proven holocarboxylase synthetase deficiency, the mean biotinyl-AMP synthetase activity at 25 nM biotin was 4% of the control mean with a range of 0.2 to 8%. This is an improved assay because it does not require preparation of an apocarboxylase substrate and is suitable for the diagnosis of patients with holocarboxylase synthetase deficiency.
Original language | English (US) |
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Pages (from-to) | 250-255 |
Number of pages | 6 |
Journal | Molecular genetics and metabolism |
Volume | 64 |
Issue number | 4 |
DOIs | |
State | Published - Aug 1998 |
Externally published | Yes |
Keywords
- Biotin
- Holocarboxylase synthetase deficiency
- Multiple carboxylase deficiency
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Biochemistry
- Molecular Biology
- Genetics
- Endocrinology