TY - JOUR
T1 - 3-Hydroxy-3-methylglutaric aciduria
T2 - a new assay of 3-hydroxy-3-methylglutaryl-CoA lyase using high performance liquid chromatography
AU - Gibson, Kenneth M.
AU - Sweetman, Lawrence
AU - Nyhan, William L.
AU - Page, Theodore M.
AU - Greene, Carol
AU - Cann, Howard M.
N1 - Funding Information:
This work was supported in part by US Public Health Services Grant No. HD-04608 from the National Institute of Child Health and Human Development, and Grant No. GM-17702 from the National Institute of General Medical Sciences, National Institutes of Health Bethesda, MD, USA.
PY - 1982/12/9
Y1 - 1982/12/9
N2 - A new assay has been developed for 3-hydroxy-3-methylglutaryl-CoA lyase, the final enzyme in the leucine degradative pathway. The assay was performed by incubating lysates of fibroblasts with [glutaryl-3-14C](d,l)-3-hydroxy-3-methylglutaryl coenzyme A. The products were analysed by high performance liquid chromatography with continuous liquid scintillation counting. This provided simultaneous identification and quantification of one of the enzymatic products, [3-14C] acetoacetic acid. The mean 3-hydroxy-3-methylglutaryl-CoA lyase activity in fibroblasts from five controls was 732 ± 81 (SD) pmol/min · mg protein. Using this assay, we have studied skin fibroblasts cultured from a patient with 3-hydroxy-3-methylglutaric aciduria and found 3% of normal 3-hydroxy-3-methylglutaryl-CoA lyase activity. The activities in skin fibroblasts cultured from the parents were 46 and 53% of control activity which is consistent with heterozygocity. Kinetic studies of 3-hydroxy-3-methylglutaryl-CoA lyase in skin fibroblasts cultured from two normal subjects yielded Km values of 14.4 and 18.8 μmol/l for 3-hydroxy-3-methylglutaryl-CoA.
AB - A new assay has been developed for 3-hydroxy-3-methylglutaryl-CoA lyase, the final enzyme in the leucine degradative pathway. The assay was performed by incubating lysates of fibroblasts with [glutaryl-3-14C](d,l)-3-hydroxy-3-methylglutaryl coenzyme A. The products were analysed by high performance liquid chromatography with continuous liquid scintillation counting. This provided simultaneous identification and quantification of one of the enzymatic products, [3-14C] acetoacetic acid. The mean 3-hydroxy-3-methylglutaryl-CoA lyase activity in fibroblasts from five controls was 732 ± 81 (SD) pmol/min · mg protein. Using this assay, we have studied skin fibroblasts cultured from a patient with 3-hydroxy-3-methylglutaric aciduria and found 3% of normal 3-hydroxy-3-methylglutaryl-CoA lyase activity. The activities in skin fibroblasts cultured from the parents were 46 and 53% of control activity which is consistent with heterozygocity. Kinetic studies of 3-hydroxy-3-methylglutaryl-CoA lyase in skin fibroblasts cultured from two normal subjects yielded Km values of 14.4 and 18.8 μmol/l for 3-hydroxy-3-methylglutaryl-CoA.
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U2 - 10.1016/0009-8981(82)90033-X
DO - 10.1016/0009-8981(82)90033-X
M3 - Article
C2 - 6185253
AN - SCOPUS:0020369778
SN - 0009-8981
VL - 126
SP - 171
EP - 181
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
IS - 2
ER -