Assays of Pyruvate Dehydrogenase Complex and Pyruvate Carboxylase Activity (original) (raw)
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Annals of Neurology, 1989
Cultured skin fibroblasts were obtained from 11 children with lactic acidemia and neurological disturbances. The residual activities of pyruvate dehydrogenase complex were 9 to 45% of control values in all specimens. Immunoblot analysis of mitochondrial proteins using polyclonal antibodies against the alpha and beta subunits of the first component (El) of the pyruvate dehydrogenase complex revealed markedly decreased amounts of cross-reacting material in 4 boys who died in infancy. Two of the boys were half brothers related through a common mother. A fifth boy had an alteration of the electrophoretic mobility of the Elcu subunit and normal EIP subunit abundance. The remaining 6 patients (2 boys and 4 girls) had normal findings on Western blot assay, and all 11 patients had normal EZ and E3 patterns. These findings suggest that the Ela subunit gene represents a genetically vulnerable site on the X chromosome. Decreased abundance of El components appears to be associated with death in infancy. A normal Western blot analysis is compatible with long-term survival despite decreased catalytic activity of the pyruvate dehydrogenase complex. Old SE, De Vivo DC. Pyruvate dehydrogenase complex deficiency: biochemical and immunoblot analysis of cultured skin fibroblasts. Ann Neurol 1989;26:746-75 1 From the
Systemic Deficiency of the First Component of the Pyruvate Dehydrogenase Complex
Pediatric Research, 1987
An infant with lactic acidosis and developmental delay had neuropathological changes consistent with Leigh's necrotizing encephalomyelopathy. Total pyruvate dehydrogenase complex (PDC) activity was low relative to controls in lymphocytes (0.2 versus 1.9 2 0.6 S D nmol/min/mg protein) and cultured skin fibroblasts (0.9 versus 2.7 + 1.0). Liver, muscle, heart, and kidney mitochondria oxidized several substrates normally, but did not oxidize pyruvate. PDC activity was absent in these mitochondria (0.1 versus 9.8 + 4.2 in liver and 0.7 versus 75 + 26 in muscle) and was very low in all tissue homogenates. Activity of the first component was low in liver mitochondria, whereas activities of the second and third components were normal. Western blot analysis of tissue proteins showed normal amounts of second and third component of PDC but undetectable to trace amounts of both a and B subunits of the first component of PDC in liver, brain, kidney, heart, and skin fibroblasts. Thus, profound systemic deficiency of PDC was due to lack of both subunit proteins of the first component of PDC.
Immunological and Biosynthetic Studies of the Human Pyruvate Dehydrogenase Complex
2007
The human pyruvate dehydrogenase multi-enzyme complex (PDC) catalyses the oxidative decarboxylation of pyruvate, transferring the resultant acetyl group to coenzyme A. It belongs to the family of 2-oxoacid dehydrogenase complexes that includes the 2-oxoglutarate dehydrogenase (OGDC) and branched-chain 2-oxoacid dehydrogenase complexes (BCOADC). Each assembly consists of multiple copies of three distinct component enzymes termed E1, E2 and E3. Human PDC also contains an accessory subunit (E3BP) that mediates stable E3 integration into the E2 'core' of the complex. Human E2-PDC consists of the following domains: two tandemly-repeated, 3.3.2.1.2 Mutation of single conserved N-and C-terminal residues located outside the central block differing in LD-OGDC.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992
... We also thank Kimberly Motter for her technical assistance. References 1 Koike, M., Reed, L. and Carroll, W. (1960) J. Biol. Chem. 235, 1924-1940. 2 DeVivo, D., Haymond, M., Obert, K., Nelson, J. and Pagliara, A. (1979) Ann. Neuro]. 6., 483-494. ...
Immunochemical evidence of pyruvate dehydrogenase (E1) deficiency
Journal of Inherited Metabolic Disease, 1988
Mammalian pyruvate dehydrogenase complex (PDHC) is a multienzyme complex with a total Mr of approximately 7 × 106. It consists of five components: pyruvate dehydrogenase (El; EC 1.2.4.1), dihydrolipoyl transacetylase (E2; EC 2.3.1.12), lipoamide dehydrogenase (E3; EC 1.6.4.3) and two specific regulatory enzymes. E1 consists of two different peptides, EI~ and EI~. An additional component of PDHC, protein X, has been elucidated immunochemically. PDHC deficiency is a rare disease and is a cause of primary lactic acidosis. While there are many reports of this disease, there are few analyses of its molecular basis in the literature. We report here our immunochemical analyses of PDHC in skin fibroblasts and/or Epstein-Barr transformed lymphoid cells obtained from three patients with this disease. MATERIALS AND METHODS Case 1: The proband was a 1-year-old girl delivered by cesarian section at a full term. Ventricular dilatation in the fetus was detected by ultrasound. At birth, there was a slight cyanosis, her cry and muscle tonus were weak and her fingers were contracted. Brain CT scan showed ventricular dilatation and agenesis of the corpus callosum. Serum lactate and pyruvate were greatly elevated, the values being lactate 112.9mg/dl and pyruvate 8.6mg/dl. Case 2: This patient was a 9-year-old girl born after a full-term pregnancy and spontaneous delivery. Tube feeding was required because of a poor sucking ability from birth on. Delay in development was prominent at the age of 3 months when she had no visual response or head control. At 6 months, she was prescribed anticonvulsant drug therapy for infantile myoclonic seizures which had become difficult to control. Brain CT scan showed a diffuse brain atrophy. The levels of lactate (41.2mg/dl) and pyruvate (3.9mg/dl) were elevated.