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.
Morita
J
J
Department of Pediatrics, University of California, San Diego, La Jolla, California 92093, USA.
Thuy
L P
LP
Sweetman
L
L
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