Akito Kitano

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.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complexes of lymphoblastoid cell lines derived from patients with classical maple syrup urine disease (MSUD) phenotypes were studied in terms of their catalytic functions and analyzed by immunoblotting, using affinity purified anti-bovine BCKDH antibody. Kinetic studies on three cell lines derived from patients with the classical phenotype showed sigmoidal or near sigmoidal kinetics for overall BCKDH activity and a deficiency of the E1 component activity. An immunoblot study revealed a markedly decreased amount of the E1 beta subunit accompanied by weak staining of the E1 alpha subunit. The E2 and E3 component exhibited a cross-reactive peptide. Thus, in at least some patients with MSUD, mutations of the E1 beta subunit might provide an explanation for the altered kinetic properties of the BCKDH complex.

Four-hydroxyphenylpyruvic acid oxidase deficiency with normal fumarylacetoacetase: a new variant form of hereditary hypertyrosinemia.

Summary: Enzymatic studies on the liver of an infant are described-a case of hypertyrosinemia without hepatic dysfunction. His parents were siblings and the mother had hypertyrosinemia. Excessive amounts of 4-hydroxyphenylpyruvic acid (pHPP), 4-hydroxyphenylacetic acid (pHPL), and 4-hydroxyphenylacetic acid (pHPA) were found to be excreted in the patients urine as well as in the urine of the mother and the inhibitor of porphobilinogen synthetase was not found. Soluble tyrosine aminotransferase (s-TAT), separated from that of the mitochondrial form (m-TAT) by DE 52 column chromatography, was normal in the patients liver, both quantitatively and qualitatively. The activities of fumarylacetoacetase in the patients liver and in the peripheral leucocytes from the parents were normal. The activity of pHPP oxidase in the patients liver was approximately 5% of the control and the enzyme had a high Km value for pHPP (controls: 0.06 ± 0.01 mM, patient: 0.23 ± 0.03 mM). From these results, the patient was thought to be different from previously described types of tyrosinemia and perhaps representative of a new variant form.This is the first report concerning 4-hydroxyphenylpyruvic acid oxidase deficiency alone. Mild metal retardation and mild hypertyrosinemia may be offered as typical clinical features of the disease.

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last step of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for defects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA in cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.

Biochemical basis of prolidase deficiency. Polypeptide and RNA phenotypes and the relation to clinical phenotypes.

Cultured skin fibroblasts or lymphoblastoid cells from eight patients with clinical symptoms of prolidase deficiency were analyzed in terms of enzyme activity, presence of material crossreacting with specific antibodies, biosynthesis of the polypeptide, and mRNA corresponding to the enzyme. There are at least two enzymes that hydrolyze imidodipeptides in these cells and these two enzymes could be separated by an immunochemical procedure. The specific assay for prolidase showed that the enzyme activity was virtually absent in six cell strains and was markedly reduced in two (less than 3% of controls). The activities of the labile enzyme that did not immunoprecipitate with the anti-prolidase antibody were decreased in the cells (30-60% of controls). Cell strains with residual activities of prolidase had immunological polypeptides crossreacting with a Mr 56,000, similar to findings in the normal enzyme. The polypeptide biosynthesis in these cells and the controls was similar. Northern blot analyses revealed the presence of mRNA in the polypeptide-positive cells, yet it was absent in the polypeptide-negative cells. The substrate specificities analyzed in the partially purified enzymes from the polypeptide-positive cell strains differed, presumably due to different mutations. Thus, there seems to be a molecular heterogeneity in prolidase deficiency. There was no apparent relation between the clinical symptoms and the biochemical phenotypes, except that mental retardation was present in the polypeptide-negative patients. The activities of the labile enzyme may not be a major factor in modifying the clinical symptoms.

Mitochondrial cytopathy with lactic acidosis, carnitine deficiency and DeToni-Fanconi-Debré syndrome

We reported a 6-year-old girl with mitochondrial cytopathy with lactic acidosis. The patient developed hypotonia, hearing loss, mental retardation, short stature, cataracta, hypoparathyroidism, DeToni-Fanconi-Debré syndrome and carnitine deficiency. Histological examination disclosed ragged red fibers and moderate lipid storage in skeletal muscle tissue and several structural abnormalities of mitochondria both in muscle tissue and proximal renal tubules. Biochemical examination of muscle tissue revealed a partial deficiency of pyruvate dehydrogenase complex and normal activities of cytochrome c oxidase, succinate cytochrome c reductase and NADH cytochrome c reductase. This is the first report of mitochondrial cytopathy representing DeToni-Fanconi-Debré syndrome associated with partial deficiency of pyruvate dehydrogenase complex and normal cytochrome c oxidase activity.

Characterization of a point mutation in the pyruvate dehydrogenase E1α gene from two boys with primary lactic acidaemia

SummaryWe report here a novel mutation in the codon for amino acid 263 resulting in the change from arginine to glutamine in the pyruvate dehydrogenase (PDH) E1α gene, in two boys with primary lactic acidaemia, from independent families. The mutation changes an amino acid located between the two serine residues which are the sites of phosphorylation of the subunit protein. In one family, the mutation wasde novo and in the other it was transmitted from mother to son. The amino acid substitution may affect function of the PDH complex via phosphorylation and dephosphorylation of the E1α subunit. Derangement in the regulation of activity of the PDH complex may explain the primary lactic acidaemia in the patients.

Mutation of the 97-197-197-1subunit of the pyruvate dehydrogenase complex, in relation to heterogeneity

SummaryPyruvate dehydrogenase complex was studied using bio- and immunochemical methods in cultured cells derived from two patients with the severe type and one patient with the mild type of pyruvate dehydrogenase complex deficiency. In patients 1 and 2, enzyme activity was all but undetectable and associated with the absence of E1α subunit of the complex. Patient 3 had a slightly reduced level of enzyme activity, and this was associated with a larger form of E1α subunit. The amount and size of E1α mRNA in the three patients was similar to that of control samples. Thus, the severity of E1α deficiency in these three patients is likely to depend on the type of mutation in the pyruvate dehydrogenase E1α subunit and the synthesis and degradation rate of the subunit.

Deduced amino acid sequence of human prolidase and molecular analyses of prolidase deficiency

Prolidase (peptidase D, imidodipeptidase, EC 3.4.13.9) splits dipeptides with a prolyl residue at the carboxy-terminal position. Deficiency of prolidase (McKusick 26413) is an autosomal recessive disorder characterized by mental retardation, various skin lesions and abnormalities of collagenous tissues. The nucleotide sequence of the cDNA inserts and partial amino acid sequence of the peptides allow us to deduce the amino acid sequence of the subunit protein of human prolidase. The cDNA inserts are used for Northern blot analyses of mRNA obtained from patients with prolidase deficiency

Immunochemical analysis of pyruvate dehydrogenase complex in 2 boys with primary lactic acidemia

We examined the pyruvate dehydrogenase (PDH) complex using bio- and immunochemical methods with cultured cells derived from 2 boys with mental retardation, ataxia, and primary lactic acidemia due to partial deficiency in the PDH complex. We found a defect in dephosphorylation and the subsequent activation of the E1± subunit of the enzyme.

Zinc status of untreated histidinemic children

Summary: In order to study zinc status in histidinemia, serum and hair zinc cocentrations were measured in 40 untreated children with histidinemia (age 2 months-5 years). In 20 children (> 2 years of age) zinc content and carbonic anhydrase activity of erythrocytes and urinary excretion of zinc were also studied. The amount of zinc excreted was elevated in histidinemic children and showed a positive correlation with the urinary histidine concentration (γ = 0.57, p < 0.005). The means of serum zinc concentration, erythrocyte zinc concentration, and erythrocyte carbonic anhydrase activity were all similar in the histidinemic and the control children. Hair zinc concentration of histidinemic children was compared with that of controls of five different age groups: <5 months, 5–18 months, 18 months-2 years, 2–4 years, and 4–5 years. In all of these age groups, hair zinc content was similar. The incidence of low-hair-zinc level (<80 μg/g) in histidinemic children >5 months of age (9 of 34) was significantly higher than in controls (18 of 180, p < 0.05). The observation suggested the possibility that untreated histidinemia may cause chronic mild zinc deficiency in some histidinemic children.