Further Analysis of Mutant Thiolase Protein in Fibroblasts from a Japanese Boy with 3-Ketothiolase Deficiency.

Abstract

We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulselabeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father ; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty.3-ktothiolase deficiency ; inherited metabolic disorder ; organic acidemia ; mitochondrial acetoacetyl-CoA thiolase ; immunochemical study 3-Ketothiolase deficiency (3KTD) is an inherited metabolic disorder of organic acids, showing autosomal recessive traits. It is characterized by hyperexcretion of 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate and tiglylglycine in

Beta-Ketothiolase Deficiency

“…To our knowledge, apart from the common R208X mutation that has been identified in Vietnamese cases, no other common mutations have been determined. 1,4,7 -15,17,20 -25,28,29,33 -59…”

Biochemical and Immunochemical Study of Seven Families with 3-Ketothiolase Deficiency: Diagnosis of Heterozygotes Using Immunochemical Determination of the Ratio of Mitochondrial Acetoacetyl-CoA Thiolase and 3-Ketoacyl-CoA Thiolase Proteins

“…It was difficult to identify unequivocally heterozygotes when immunoblot analysis was performed using anti-T2 antibody alone (I 1). However, we observed that the identification of carriers could be convincingly achieved by performing immunoblot analysis using a mixture of anti-T2 and anti-T1 antibodies, with the T1 band serving as an internal standard (15,16,19,20).…”