J. Morita et al., DEFICIENCY OF BIOTINYL-AMP SYNTHETASE-ACTIVITY IN FIBROBLASTS OF PATIENTS WITH HOLOCARBOXYLASE SYNTHETASE DEFICIENCY, MOLECULAR GENETICS AND METABOLISM, 64(4), 1998, pp. 250-255
Citations number
19
Categorie Soggetti
Genetics & Heredity","Medicine, Research & Experimental",Biology
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.