Search PubMed⌕ Search

Biomedical subjects

T Saheki

Publications and source records attributed to T Saheki.

At least 145 records · Page 8Linked to original sources
PubMed1986

Physical and functional association of the atrial natriuretic peptide receptor with particulate guanylate cyclase as demonstrated using detergent extracts of bovine lung membranes.

Coupling of the atrial natriuretic peptide (ANP) receptor to particulate guanylate cyclase has been demonstrated kinetically and chromatographically using bovine lung plasma membranes and their detergent extracts. Addition of ANP to the membrane suspension stimulated guanylate cyclase activity 2-5-fold indicating the presence of ANP-sensitive particulate guanylate cyclase. The enzyme retained the ability to respond to ANP even after solubilization with digitonin. Characterization of the solubilized enzyme by gel filtration and affinity chromatography revealed that the ANP receptor and particulate guanylate cyclase exist as a functionally but not covalently linked stable complex.

Animals↗
PubMed1986

Absence of argininosuccinate lyase protein in the liver of two patients with argininosuccinic aciduria.

The enzyme defects in two cases of argininosuccinic aciduria were examined at the molecular level by enzymatic and immunological methods. No argininosuccinate lyase activity was detected in the liver or erythrocytes of either patient nor in the kidney or brain of one of the patients even in the presence of high concentrations of the substrate. The titration curve of antiserum to human argininosuccinate lyase with the liver extract from a control subject was not affected by the addition of the liver extracts from one of the patients. Double immunodiffusion analysis revealed a single precipitin line between the purified antiserum and the liver extract from a control, but no precipitin lines between the antisera and the liver extracts from the two patients. These results indicate a complete or almost complete defect of an immunologically cross-reactive material in the liver of the patients.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver.

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.

Animals↗
PubMed1986

Characterization and immunochemistry of pyruvate dehydrogenase complex of Ascaris muscle.

Pyruvate dehydrogenase complex and lipoamide dehydrogenase were purified from muscle of Ascaris lumbricoides var. suum which contains relatively a large amount of the complex. Molecular weights of three constituent enzymes of Ascaris pyruvate dehydrogenase complex were as follows; alpha- and beta-subunits of pyruvate dehydrogenase were 42,000 and 37,000, respectively, lipoate acetyltransferase was 76,000 and lipoamide dehydrogenase was 56,000. Furthermore, two unknown polypeptides having molecular weight of 46,000 and 41,000 were detected. Anti-Ascaris lipoamide dehydrogenase antibody precipitated three constituent enzymes and two unknown polypeptides, suggesting that lipoamide dehydrogenase not only binds tightly to complex, but also two unknown polypeptides bind tightly to complex.

Animals↗
PubMed1986

Messenger RNA coding for argininosuccinate synthetase in citrullinemia.

Messenger RNA coding for argininosuccinate synthetase (ASS), extracted from the livers of some patients with citrullinemia, was analyzed using a cell-free translation system and dot and Northern blot hybridization with cDNA probe for ASS. In patients with quantitative-type citrullinemia, called type II here, previous studies have demonstrated that the hepatic content of the enzyme was about 10% of the control value, whereas the translatable mRNA level for the enzyme was similar to that of control livers. Here, we confirmed that the type II liver contained an almost normal amount of mRNA coding for ASS, judged by the dot-blot hybridization technique with cDNA. Northern blot hybridization of RNA indicated that there was hybridizable mRNA of approximately normal size (about 1.7 kilobase [kb]) in each, suggesting that large structural gene deletions had not occurred. These results indicate that in type II citrullinemia, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased or inhibited translation in the liver. Another type of citrullinemia was found and classified as type III. It is characterized by no detectable enzyme activity for ASS or translation activity for ASS mRNA. However, a smaller amount of RNA molecule hybridized for ASS cDNA was detected.

Adult↗
PubMed1986

Molecular cloning of cDNA for argininosuccinate lyase of rat liver.

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.

Animals↗
PubMed1985

Immunochemical comparison of lipoamide dehydrogenases from various sources and reactivity of various lipoamide dehydrogenases with rat heart pyruvate dehydrogenase-subcomplex.

Lipoamide dehydrogenases from various sources were purified and their immunochemical properties were compared. Antibody against rat lipoamide dehydrogenase reacted with rat, human, pig, pigeon and frog enzymes, but not with enzymes from E. coli, yeast and Ascaris. Anti-Ascaris enzyme and anti-E. coli enzyme antibodies reacted with Ascaris and E. coli enzymes, respectively. The pyruvate dehydrogenase subcomplex, which consists of pyruvate dehydrogenase and lipoate acetyltransferase, was prepared by releasing the lipoamide dehydrogenase from rat heart pyruvate dehydrogenase complex by anti-lipoamide dehydrogenase antibody. Lipoamide dehydrogenases from various sources were added to rat pyruvate dehydrogenase subcomplex and the complex overall activity was measured. Each lipoamide dehydrogenase effectively recovered the overall activity of rat pyruvate dehydrogenase subcomplex to 80% of the original activity.

Animals↗
PubMed1985

Level of translatable messenger RNA coding for argininosuccinate synthetase in the liver of the patients with quantitative-type citrullinemia.

The translation activity of mRNA coding for argininosuccinate synthetase in total RNA extracted from the liver of three patients with quantitative-type citrullinemia was determined using a cell-free translation system. In two patients, the hepatic content of the enzyme was about 20% of the control value, whereas translatable mRNA level for the enzyme was similar to or slightly lower than those of control livers. In the third patient, the enzyme content was about 50% of the control value, and mRNA activity for the enzyme was low normal. These results indicate that at least in the first two patients, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased translation in the patient's liver.

Adult↗
PubMed1985

Transient hyperammonemia during aging in ornithine transcarbamylase-deficient, sparse-fur mice.

Ornithine transcarbamylase [EC 2.1.3.3] (OTC) deficient, sparse-fur (spf) mice are considered to be a good model of human OTC deficiencies. We measured the blood ammonia concentration in spf mice. It was not always at a high level. It was high only during weaning, then gradually decreased and finally fell to a normal level. A similar phenomenon was observed in the change of hepatic ammonia concentration. Excretion of urinary orotate in spf mice was 15-20 times higher than in control mice just after weaning. It also gradually decreased during aging, but remained at a high level compared with adult control mice. OTC activity at pH 7.7 in the liver of spf mice increased significantly between the weaning period and 30-40 days old, when blood ammonia was falling to a normal level.

Aging↗
PubMed1984

Pyruvate dehydrogenase subcomplex with lipoamide dehydrogenase deficiency in a patient with lactic acidosis and branched chain ketoaciduria.

The overall and three component activities of pyruvate dehydrogenase complex were measured in the liver and muscle from a patient who died at 1.9 years with increased concentrations of serum lactate, alpha-ketoglutarate and branched chain amino acids. The component activities of both lipoate acetyltransferase and pyruvate dehydrogenase were similar to those of normal controls, but the overall pyruvate dehydrogenase complex activity was 11 to 30% of controls and lipoamide dehydrogenase activity was not detected. The overall activity was significantly increased by the addition of lipoamide dehydrogenase purified from human liver. Immunochemical studies carried out with antibody prepared against lipoamide dehydrogenase from rat liver, could detect no immunoreactive material in liver and muscle homogenates from the patient, suggesting that the deficiency of lipoamide dehydrogenase activity was due to the lack of enzyme protein.

Acetyltransferases↗
PubMed1984

Molecular basis of ornithine transcarbamylase deficiency lacking enzyme protein.

We report an ornithine transcarbamylase(OTC)-deficient male patient who had no detectable immunoreactive materials but did have active mRNA for OTC-related protein. The total absence of OTC activity in the liver of the patient was caused by a complete lack of immunoreactive material, as determined by Ouchterlony double immunodiffusion, single radial immunodiffusion, and sodium dodecylsulphate-polyacrylamide gel electrophoresis of immunoprecipitate and of liver homogenate. However, mRNA coding for the precursor of OTC was clearly detected in autopsy specimens of the patient's liver as well as of controls in a cell-free translation system consisting of rabbit reticulocyte lysates and [35S]methionine. The labelled precursor of OTC synthesized in vitro with mRNA from the patient could be transported into rat liver and kidney mitochondria and processed to form a protein with a molecular weight indistinguishable from mature OTC, suggesting that there was no defect in the protein structure necessary for its transport into mitochondria. These results suggest that the primary defect of the OTC deficiency was located in the structural gene and that the labile OTC-related protein, after being synthesized with its mRNA, was degraded too rapidly to be detected by the method used.

Amino Acid Metabolism, Inborn Errors↗
PubMed1983

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex.

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

Animals↗
PubMed1983

Kinetic analysis of argininosuccinate synthetase in a variant form of citrullinaemia.

Argininosuccinate (ASA) synthetase in the liver from a patient with a variant form of citrullinaemia was analysed. Serum citrulline level was approximately 40 times higher than the control level, and the ASA synthetase activity was approximately 10% that of the control. Other urea cycle enzymes were within normal ranges. The specific activity (enzyme activity/enzyme protein, measured by immunochemical method), pH optimum, Michaelis constants, and thermal denaturation in the presence or absence of ASA were similar to those in enzymes obtained from the normal and the patient's liver cells. No in vitro effect of the patient's liver homogenate on ASA synthetase in normal liver was observed. Serum citrulline levels of the patient's parents and young brother were at the maximal level of control at fasting and definitely elevated 4 h after citrulline loading (100 mg/kg).

Adult↗