Deficiency of biotinyl-AMP synthetase activity in fibroblasts of patients with holocarboxylase synthetase deficiency.
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.
Department of Pediatrics, University of California, San Diego, La Jolla, California 92093, USA.
J
J
Morita
L P
LP
Thuy
L
L
Sweetman
D019731:Carbon-Nitrogen Ligases / Q000172:deficiency* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D007231:Infant, Newborn; D007700:Kinetics; D008297:Male; D008661:Metabolism, Inborn Errors / Q000201:enzymology
D019731:Carbon-Nitrogen Ligases; C115475:biotinyl-AMP synthetase; C023312:holocarboxylase synthetases