Altered kinetic properties of the branched-chain alpha-keto acid dehydrogenase complex due to mutation of the beta-subunit of the branched-chain alpha-keto acid decarboxylase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.

Indo, Yasuhiro; Kitano, Akihiko; Endo, Fumio; Akaboshi, Izumi; Matsuda, Ichiro

doi:10.1172/jci113064

Cited by 48 publications

(25 citation statements)

References 43 publications

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“…The Vm. and apparent Km values were [13][14][15] nmol/h per milligram ofprotein and 0.053-0.056 mM, respectively. Cells from the patient (T.H.)…”

Section: Resultsmentioning

confidence: 94%

“…We (13,14) and others (15) reported that some patients with MSUD showed decreased amounts of E2 protein. To investigate related molecular mechanisms, we and others isolated a cDNA clone encoding the entire E2 subunit of the human BCKDH complex (16,17).…”

Section: Introductionmentioning

confidence: 77%

“…and apparent Km in the patient were 1.2 nmol/h per milligram of protein and 0.103 mM, respectively. Another cell strain related to E2 deficiency (GM 1366) was analyzed as a reference (13,14) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Substrate-dependent kinetics of the BCKDH was determined for disrupted lymphoblastoid cells by quantifying the '4CO2 released from a-keto [1-'4C] isovaleric acid, in the presence of co-factors, as described ( 13,14). Immunoblot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Mitochondrial proteins were resolved by electrophoresis in a 10% polyacrylamide gel in the presence of SDS essentially as described by Laemmli (19). The resolved proteins were electroblotted to nitrocellulose and cross-reacting proteins were detected using affinity-purified anti-bovine BCKDH (E, + E2) rabbit immunoglobulin, as described (13,14). Analysis ofcDNA.…”

Section: Methodsmentioning

confidence: 99%

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Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

Mitsubuchi¹,

Nobukuni²,

Akaboshi³

et al. 1991

J. Clin. Invest.

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We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain a-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (M, 52,000) was absent and another protein band with an M, of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (M, 49,000) detected in the patient's cells by immunoblot analysis.Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result ofthe mutation, part ofthe inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and a-ketoglutalate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity.

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“…The Vm. and apparent Km values were [13][14][15] nmol/h per milligram ofprotein and 0.053-0.056 mM, respectively. Cells from the patient (T.H.)…”

Section: Resultsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

Mitsubuchi¹,

Nobukuni²,

Akaboshi³

et al. 1991

J. Clin. Invest.

Self Cite

View full text Add to dashboard Cite

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A two-dimensional electrophoretic map of human mitochondrial proteins from immortalized lymphoblastoid cell lines: A prerequisite to study mitochondrial disorders in patients

et al. 2005

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Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30% of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2-DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of beta-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level.

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Molecular defects of the branched-chain α-keto acid dehydrogenase complex: Maple syrup urine disease due to mutations of the E1α or E1β subunit gene

Indo

Matsuda

1996

Alpha-Keto Acid Dehydrogenase Complexes

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Altered kinetic properties of the branched-chain alpha-keto acid dehydrogenase complex due to mutation of the beta-subunit of the branched-chain alpha-keto acid decarboxylase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.

Cited by 48 publications

References 43 publications

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

A two-dimensional electrophoretic map of human mitochondrial proteins from immortalized lymphoblastoid cell lines: A prerequisite to study mitochondrial disorders in patients

Molecular defects of the branched-chain α-keto acid dehydrogenase complex: Maple syrup urine disease due to mutations of the E1α or E1β subunit gene

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