A coupled assay detecting defects in fibroblast isoleucine degradation distal to enoyl-CoA hydratase: application to 3-oxothiolase deficiency.
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.
Gibson
K M
KM
Kimberly H. Courtwright and Joseph W. Summers Metabolic Disease Center, Baylor University Medical Center, Dallas, Texas 75226.
Lee
C F
CF
Kamali
V
V
Søvik
O
O
D000101:Acetyl-CoA C-Acetyltransferase / Q000172:deficiency*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D007532:Isoleucine / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017693:Sodium Bicarbonate
D000214:Acyl Coenzyme A; D001639:Bicarbonates; D002250:Carbon Radioisotopes; D015636:Magnesium Chloride; D007532:Isoleucine; D009243:NAD; C015357:methylmalonyl-coenzyme A; C009061:propionyl-coenzyme A; C033618:tiglyl-coenzyme A; D000255:Adenosine Triphosphate; D017693:Sodium Bicarbonate; D012964:Sodium; D000101:Acetyl-CoA C-Acetyltransferase; D004746:Enoyl-CoA Hydratase