Abstract
There are five known thiolases in human fibroblasts, and all but mitochondrial trifunctional protein (TFP) have thiolase activity toward acetoacetyl-CoA (AACoA). We investigated the contribution of mitochondrial acetoacetyl-CoA thiolase (AACoAT) (T2), cytosolic AACoAT (CT), and mitochondrial 3-ketoacyl-CoA thiolase (T1) to the total AACoAT activity in control human fibroblasts. Immunotitration of AACoAT activity with antibodies against T2, CT, or T1 was carried out in control fibroblasts, with the following results. In the case of AACoAT activity in the absence of potassium ion, 26-38%, 40-47%, and 11-20% of the total activity derived from CT, T1, and T2, respectively. The residual 6-9% total activity was not immunotitrated when three antibodies were used in combination. Hence, the contribution of peroxisomal 3-ketoacyl-CoA thiolase to the total AACoAT activity in the absence of potassium ion was at least less than 6-9%. Because the normal range of total AACoAT activity is relatively wide, it is difficult to evaluate CT defects based on a decrease of total AACoAT activity. Immunotitration with anti-CT antibody in six control fibroblasts revealed that CT activity ranges between 1.3 and 2.4 nmol/min/mg of protein. Immunotitration proved to be an accurate method to evaluate CT activity. The two cell lines from patients with CT deficiency have become extinct.
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Abbreviations
- CT:
-
cytosolic acetoacetyl-CoA thiolase
- T2:
-
mitochondrial acetoacetyl-CoA thiolase
- T1:
-
mitochondrial 3-ketoacyl-CoA thiolase
- PT:
-
peroxisomal 3-ketoacyl-CoA thiolase
- TFP:
-
trifunctional protein
- AACoAT:
-
acetoacetyl-CoA thiolase
- AACoA:
-
acetoacetyl-CoA
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Acknowledgements
We thank Drs. M. J. Bennett and C. J. De Groot for their efforts in providing the CT-deficient cell lines and M. Ohara for comments on the manuscript.
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Supported in part by Grants-in-Aid for Scientific Research (07770569, 07670865) from the Ministry of Education, Science, and Culture of Japan, by a grant (5A-6-02) from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan, and by a grant from The Naito Foundation, and by a grant from the Uehara Memorial Foundation.
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Fukao, T., Song, XQ., Yamaguchi, S. et al. Immunotitration Analysis of Cytosolic Acetoacetyl-Coenzyme A Thiolase Activity in Human Fibroblasts. Pediatr Res 39, 1055–1058 (1996). https://doi.org/10.1203/00006450-199606000-00020
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DOI: https://doi.org/10.1203/00006450-199606000-00020
