[Citrullinemia].
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Amino Acid Metabolism, Inborn Errors↗
Biomedical subjects
T Saheki
Publications and source records attributed to T Saheki.
[Argininosuccinic aciduria].
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Amino Acid Metabolism, Inborn Errors↗
Immunological identification of a new component of Ascaris pyruvate dehydrogenase complex.
The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.
Animals↗
Identification of the primary translation product of atrial natriuretic peptide receptor mRNA in a cell-free system using anti-receptor antiserum.
The primary translation product of mRNA encoding atrial natriuretic peptide (ANP) receptor has been shown to have an Mr of 58,000. Poly(A)+ RNA was isolated from the bovine kidney and lung and translated in a rabbit reticulocyte lysate system containing [35S]methionine. Immunoprecipitation of the labeled translation products, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, identified a 58-kDa protein as the primary translation product which is the unglycosylated precursor to be processed to the glycosylated mature 70-kDa form found in the plasma membranes. The result lends strong support to our previous proposal that mature ANP receptor is composed of two disulfide-linked 70-kDa subunits, eliminating the possibility that the two 70-kDa subunits arise from a larger 140-kDa precursor by proteolytic cleavage.
Animals↗
Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase.
Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.
Amino Acid Sequence↗
Clinical application of enzyme immunoassay in the analysis of citrullinemia.
We have developed a sensitive enzyme immunoassay (EIA) for the quantification of argininosuccinate synthetase (ASS) in the range of 0.05-1 ng/tube using a Fab'-peroxidase conjugate prepared with maleimide compound devised by Ishikawa et al. Amounts of hepatic ASS in control subjects were determined by this method with the purified enzyme as a standard and their specific activities were calculated to be 1.3-1.4 mumol product/min (U)/mg of ASS, which was quite similar to that of the purified enzyme. Amounts of ASS in the liver of patients with three types of citrullinemia, type I, II and III according to our arbitrary classification, were determined by the EIA method. The following results were obtained: hepatic ASS from a patient diagnosed as type I citrullinemia because of its abnormally large Km values for citrulline and aspartate was calculated to have a specific activity of 0.18 U/mg of ASS, indicating that the enzyme is a variant; specific activities of ASS in the hepatic extract of type II citrullinemic patients were 1.2-1.3 U/mg of ASS, confirming the finding obtained by single radial immunodiffusion method that the decrease of the activity in type II is caused by a decrease in the total amount of ASS protein; a very small amount of immune cross-reactive material was detected in the liver of a patient diagnosed as type III citrullinemia based on findings of undetectable ASS activity in the liver and cultured skin fibroblast.
Adult↗
Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure.
The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.
Animals↗
Molecular cloning of cDNA for rat liver lipoate acetyltransferase. A component of pyruvate dehydrogenase complex.
One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector lambda gt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified tha cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.
Acetyltransferases↗
Structure of an abnormal messenger RNA for argininosuccinate synthetase in citrullinemia.
The structural abnormality of mRNA for argininosuccinate synthetase (ASS) and the structure of immune cross-reactive material for ASS (ASS-CRM) in the liver of a patient with type III citrullinemia were analyzed using dot and Northern blot hybridization, S1 nuclease analysis, and a sensitive enzyme-linked immunosorbent assay. The patient's liver contained no detectable ASS activity, the ASS-CRM content was about 0.5% of that in the control and the ASS mRNA content was almost normal. The ASS mRNA of the case was found to have an apparent structural abnormality; approximately 1.57 kb in length caused by a defect of about 0.1 kb near the 3' end of the coding region. Furthermore, gel filtration analysis revealed that the ASS-CRM had a molecular weight indistinguishable from the control ASS. These results indicate that the abnormal mRNA of the patient was translated in vivo to an immune-cross-reactive protein that was able to form a quaternary structure.
Amino Acid Metabolism, Inborn Errors↗
Abnormality of citrulline synthesis in liver mitochondria from patients with hyperornithinaemia, hyperammonaemia and homocitrullinuria.
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Amino Acid Metabolism, Inborn Errors↗
Hereditary disorders of the urea cycle in man: biochemical and molecular approaches.
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Amino Acid Metabolism, Inborn Errors↗
Increased urinary excretion of argininosuccinate in type II citrullinemia.
Although argininosuccinate is a product of the catalytic action of deficient argininosuccinate synthetase in citrullinemia, its concentration was found to be elevated in the urine of patients with type II citrullinemia. Urinary argininosuccinate was identified by two methods; its conversions to anhydride by boiling in an acidic solution and to arginine by the enzymatic action of argininosuccinate lyase. Oral administration of citrulline to patients with type II citrullinemia and control subjects increased urinary argininosuccinate levels. These phenomena are consistent with our previous findings on type II citrullinemia (Adv Exp Med Biol 1983;153:63-76,J Clin Biochem Nutr 1986;1:129-142), namely that renal argininosuccinate synthetase which plays a role in arginine synthesis is not deficient in patients with type II citrullinemia; and that serum arginine levels in patients with type II citrullinemia are rather higher than the controls, and increase after the oral administration of citrulline. The organ-specific deficiency of argininosuccinate synthetase in type II citrullinemia is further confirmed by this paper.
Administration, Oral↗
Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state.
Sparse-fur mice which are deficient in ornithine transcarbamylase, the second-step enzyme in the urea cycle, were examined for hyperammonemia and its relationship with encephalopathy. We compared amino acid concentrations in the serum and brain of spf mice with those of control mice. Unlike hepatic encephalopathy we could not find marked amino acid changes in the serum of spf mice besides low levels of citrulline and arginine. But in the brain of spf mice, glutamine was increased strikingly during hyperammonemia, and a concomitant accumulation of large neutral amino acids such as tyrosine, phenylalanine, methionine, and histidine was observed. The accumulation of these large neutral amino acids in the brain was not influenced by 24-hr fasting which caused increases in branched chain amino acids in the serum. From these results, we conclude that the accumulation of the large neutral amino acid in the brain of hyperammonemic state is caused by uptake of ammonia in the brain and the subsequent accumulation of glutamine, but is not influenced by a decreased ratio of branched chain amino acids to aromatic amino acids in the serum.
Aging↗
Visualization of ANP receptor on glomeruli of bovine kidney by use of a specific antiserum.
Atrial natriuretic peptide (ANP) receptor was purified from the bovine lung by affinity chromatography and preparative gel electrophoresis. A specific antiserum was raised against the purified receptor and used to localize receptors immunohistochemically on bovine kidney sections. Intense staining was seen in glomeruli. This distribution corresponds to that of ANP binding sites revealed by previous autoradiographic analyses and matches a glomerular action of ANP suggested by physiological studies.
Animals↗
Molecular basis of enzyme abnormalities in urea cycle disorders. With special reference to citrullinemia and argininosuccinic aciduria.
This paper deals with enzymological, immunochemical and molecular genetic analyses of citrullinemia and argininosuccinic aciduria. Citrullinemia has been classified by Saheki et al. [J. inher. Metab. Dis. 8: 155-156, 1985] into three types from the properties of the deficient argininosuccinate synthetase (ASS) of the patients. Analysis of hepatic mRNA coding for ASS revealed certain characteristics in type II and III citrullinemic patients whose hepatic ASS protein was low. A newly developed enzyme-linked immunosorbent assay (ELISA) of argininosuccinate lyase (ASL) protein showed that 8 out of ten cases of argininosuccinic aciduria had no detectable ASL protein in the liver, erythrocytes, cultured skin fibroblasts or cultured amniocytes.
Amino Acid Metabolism, Inborn Errors↗
Hyperornithinemia, hyperammonemia, and homocitrullinuria: case report and biochemical study.
Two siblings with hyperornithinemia, hyperammonemia, and homocitrullinuria are reported. The clinical picture included protein intolerance, mental retardation, seizures, and stuporous episodes. One patient had cerebellar ataxia, myoclonus, convulsive seizure, and muscular weakness in both legs. Isolated liver mitochondria in the patient revealed that ornithine transport and citrulline synthesis were decreased, but urea cycle enzymes and ornithine aminotransferase were normal. Ornithine metabolism was decreased in cultured skin fibroblasts.
Adolescent↗
Immunoenzymatic studies of ornithine transcarbamylase (OTC) in liver of patients with OTC deficiency by enzyme linked immunosorbent assay (ELISA).
Antigen contents of ornithine transcarbamylase (OTC) protein in the liver of 5 patients with OTC deficiency and mutant mice were studied by ELISA. OTC activities in the liver tissues of 2 patients were 13.1% and 5.2% of the controls, respectively. The 2 patients' antigen contents of OTC protein were decreased in parallel with the enzyme activities. OTC antigen contents were not detected in the liver tissues of remaining 3 patients who had the complete type of OTC deficiency. Enzyme activities, kinetic properties and antigen contents of the OTC deficient mutant mice were the same as the results reported previously (Briand et al. 1982). The ELISA method for the assay of OTC antigen contents used in this study is more sensitive as compared with the radial immunodiffusion technique.
Animals↗
Enzyme-linked immunosorbent assay of serum argininosuccinate synthetase.
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Argininosuccinate Synthase↗