Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

“…Cells were then suspended to a final protein concentration of 1 g/L in PBS. DCA (5 mmol/L) was added to specifically inhibit the inactivating kinase (but not the activating phosphatase) of PDHc, as described previously (20 ). The suspensions were incubated at 37°C for 15 min.…”

Section: Preparation Of Crude Tissue Homogenatementioning

confidence: 99%

Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

et al. 2005

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“…This short-term effect occurs within 15 min of incubation of fibroblasts with 5 mM DCA (22). An additional increase in maximal extractable PDHC activity has also been observed in fibroblasts after 3-5 d of 5 mM DCA supplementation (14,23).…”

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confidence: 75%

Differential Effect of DCA Treatment on the Pyruvate Dehydrogenase Complex in Patients with Severe PDHC Deficiency

et al. 2003

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Dichloroacetate (DCA) is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particularly in patients with pyruvate dehydrogenase (PDHC) deficiency. Recent reports have demonstrated that the response to DCA may depend on the type of molecular abnormality. In this study, we investigated the response to DCA in various PDHC-deficient cell lines and tried to determine the mechanism involved. The effect of chronic 3-d DCA treatment on PDHC activity was assessed in two PDHC-deficient cell lines, each with a different point mutation in the E1␣ subunit gene (R378C and R88C), and one cell line in which an 8-bp tandem repeat was deleted (W383 del). Only two (R378C and R88C) of the three PDHC-deficient cell lines with very low levels of PDHC activity and unstable polypeptides were sensitive to chronic DCA treatment. In these cell lines, DCA treatment resulted in an increase in PDHC activity by 125 and 70%, respectively, with concomitant increases of 121 and 130% in steady-state levels of immunoreactive E1␣. DCA treatment reduced the turnover of the E1␣ subunit in R378C and R88C mutant cells with no significant effect on the E1␤ subunit. Chronic DCA treatment significantly improved the metabolic function of PDHC in digitonin-permeabilized R378C and R88C fibroblasts. The occurrence of DCA-sensitive mutations suggests that DCA treatment is potentially useful as an adjuvant to ketogenic and vitamin treatment in PDHC-deficient patients. PDHC deficiency is a nuclear-encoded mitochondrial disorder and a major recognized cause of neonatal encephalopathies associated with primary lactic acidosis (1). This multienzyme complex plays an important role in the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA. E1, one of the subunits of the complex, is a thiamine pyrophosphatedependent pyruvate decarboxylase. It is a tetramer composed of two ␣ and two ␤ subunits, the ␣ subunit containing the thiamine pyrophosphate binding sites. E2 is a dihydrolipoamide acetyl transferase. E3 is a dihydrolipoamide dehydrogenase, and E3BP or protein X mediates the interaction between E2 and E3. Two elements regulate the complex: an E1 kinase and a phospho-E1 phosphatase, which phosphorylate and dephosphorylate, respectively, three serine residues in the E1 ␣ subunit, resulting in the deactivation and activation, respectively, of the complex (2).Defects in the PDHC are frequently attributed to deficiencies in the E1 ␣ component (3), encoded by chromosome X (Xp22.1-22.2) (4, 5). Considerable phenotypic and allelic heterogeneity has been observed for this defect. Previous studies have reported that a decrease in the stability of the E1 ␣ immunoreactive subunit may contribute to the expression of many E1 ␣ mutations (6, 7); very few studies to distinguish mutations that impair polypeptide stability from those impairing catalytic efficiency have been carried out to date (8).DCA is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particular...

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“…For determination of the total activity, the homogenate was preincubated with partially purified preparation of a broad specificity protein phosphatase from rabbit liver (8), and 3.7 mM MnC1, for 30 min at 37"C, and then the activity was measured as described above. The DCA-activated activity of the PDH complex was measured by a modification of the method of Sheu et al (9). The harvested cells were incubated for 15 min at 37°C in PBS containing 5 mM DCA to activate PDH.…”

Section: Chemicals [I -'4cmentioning

confidence: 99%

“…The fully activated enzyme probably furnishes the most reliable estimate of enzymatic activity. The enzyme can be activated by pretreating the skin fibroblasts for 15 min with DCA, an inhibitor of PDH kinase (EC 2.7.1.99), before the cells are disrupted for measurement of activity of the PDH complex (9). For use in screening for disorders of pyruvate metabolism, we also developed a sensitive assay method for measuring the rates of decarboxylation of [l-'4C]pyruvate during in vitro culture of skin fibroblasts with DCA.…”

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confidence: 99%

Detection of Pyruvate Metabolism Disorders by Culture of Skin Fibroblasts with Dichloroacetate

et al. 1988

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ABSTRACT. For use in screening for disorders of pyruvate metabolism, a sensitive assay method was developed for measuring the rate of decarboxylation of [1-14C]pyruvate during in vitro culture of skin fibroblasts with dichloroacetate (DCA). The rate of decarboxylation of [1-14C]pyruvate by skin fibroblasts from control subjects increased from 59.6 + 13.2 to 97.3 + 12.0 nmol/h/mg protein during in vitro culture in medium supplemented with 10 mM DCA for 3 days. In contrast, the rate hardly increased in cells from four of 20 patients with congenital lactic acidosis of unknown cause during in vitro culture with DCA. On day 3 of culture, the values for the four patients did not overlap those of control cells and so these four patients could be clearly distinguished from control subjects. Measurements of the original activity and the activity of the pyruvafe dehydrogenase (PDH) complex after activation with a broad specificity protein phosphatase and DCA suggested that in three of the patients the aberration was a disorder in the mechanism for activation of PDH, including deficiency of PDH phosphatase or a mutation of PDH itself, whereas that in the fourth patient it might be a disorder of the mitochondria1 transport system for pyruvate. Thus, measurement of the rate of decarboxylation of [I-14C] pyruvate by skin fibroblasts cultured in medium supplemented with 10 mM DCA for 3 days is a useful method for screening for disorders of pyruvate metabolism in cultured skin fibroblasts. (Pediatr Res 23: [561][562][563][564] 1988) Abbreviations DCA, dichloroacetate PDH, pyruvate dehydrogenase PBS, phosphate-buffered saline Chronic congenital lactic acidosis of childhood is characterized by persistently high levels of blood pyruvate and lactate. Most patients with this disease die in infancy or show growth and psychomotor retardation because no adequate treatment is available. Recently, the number of reported cases of congenital lactic acidosis has been increasing. Hereditary deficiencies leading to lactic acidosis have been demonstrated in the enzymes of the PDH (EC 1.2.4.1) complex, in the key enzymes of the pathway of gluconeogenesis and in the enzymes of the electron transport system (I). However, in many patients with congenital lactic acidosis, the primary biochemical defect is unknown (2, 3). 56Therefore, we have been trying to develop a sensitive method for screening for disorders of pyruvate metabolism in cultured skin fibroblasts from patients with congenital lactic acidosis. Herein we found that measurement of the rate of decarboxylation of [l-14C]pyruvate during in vitro culture of the fibroblasts with DCA, which is an activator of PDH, is a useful screening method for disorders of pyruvate metabolism. We also found that for determination of the cause of PDH deficiency, both the original activity of the PDH complex and also its total activity induced by in vitro activations with a broad specificity protein phophatase and DCA must be determined. CulturedJibroblasts. Fibroblasts were grown in minimal essential ...

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Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

Cited by 140 publications

References 50 publications

Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

Differential Effect of DCA Treatment on the Pyruvate Dehydrogenase Complex in Patients with Severe PDHC Deficiency

Detection of Pyruvate Metabolism Disorders by Culture of Skin Fibroblasts with Dichloroacetate

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