DEFICIENCY OF BIOTINYL-AMP SYNTHETASE-ACTIVITY IN FIBROBLASTS OF PATIENTS WITH HOLOCARBOXYLASE SYNTHETASE DEFICIENCY

A simple, rapid assay was developed to diagnose holocarboxylase synthe tase deficiency. Holocarboxylase synthetase first catalyzes the format ion of biotinyl-AMP from biotin and ATP, an activity designated as bio tinyl-AMP synthetase. In the second step of the reaction, biotin is tr ansferred from biotinyl-AMP to the enzymatically inactive apocarboxyla se to form an active holocarboxylase. The assay for holocarboxylase sy nthetase activity therefore requires a protein apocarboxylase substrat e which is not readily available. In the assay for biotinyl-AMP synthe tase, hydroxylamine reacts nonenzymatically with the product of the en zymatic reaction, biotinyl-AMP, to form biotinylhydroxamate. At the en d of the reaction, unreacted radioactive biotin substrate, which is ne gatively charged at neutral pH, is bound to an anion-exchange resin an d a neutral radioactive biotinylhydroxamate product in the supernatant is counted. In fibroblasts from 11 patients with proven holocarboxyla se 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%. T his is an improved assay because it does not require preparation of an apocarboxylase substrate and is suitable for the diagnosis of patient s with holocarboxylase synthetase deficiency. (C) 1998 Academic Press.