A coupled assay detecting defects in fibroblast isoleucine degradation distal to enoyl-CoA hydratase: application to 3-oxothiolase deficiency.
Clin Chim Acta
; 205(1-2): 127-35, 1992 Jan 31.
Article
in En
| MEDLINE
| ID: mdl-1355701
ABSTRACT
We developed a coupled NaH14CO3 fixation assay to detect 3-oxothiolase deficiency in extracts of cultured human fibroblasts. Cell extracts were incubated with tiglyl-CoA, NAD, CoASH, ATP and NaH14CO3. The enzymatic activities of tiglyl-CoA (enoyl-CoA) hydratase, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase and 2-methylacetoacetyl-CoA thiolase (3-oxothiolase) were coupled to produce propionyl-CoA. Propionyl-CoA produced in the assay was estimated by fixation of NaH14CO3 into [14C]methylmalonyl-CoA employing endogenous propionyl-CoA carboxylase. The control activity was 32 +/- 23 pmol/min per mg protein (+/- 1 S.D., range 7-94; 28 cell lines). Five known cases of 3-oxothiolase deficiency had a mean activity of 2% of the control; a sixth case of 3-oxothiolase deficiency was significantly higher at 27% of the mean control value. Coupled assay activity was also low (3% of control) in the cells from a patient with propionyl-CoA carboxylase deficiency.
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Database:
MEDLINE
Main subject:
Acetyl-CoA C-Acetyltransferase
/
Enoyl-CoA Hydratase
/
Fibroblasts
/
Isoleucine
Limits:
Humans
Language:
En
Year:
1992
Type:
Article