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Biomedical subjects

G Kikuchi

Publications and source records attributed to G Kikuchi.

At least 73 records · Page 4Linked to original sources
PubMed1979

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.

Animals↗
PubMed1979

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.

Animals↗
PubMed1978

Reaction of the microsomal heme oxygenase with cobaltic protoporphyrin IX, and extremely poor substrate.

A reconstituted heme oxygenase system which was composed of a purified heme oxygenase from pig spleen microsomes and a partially purified NADPH-cytochrome c reductase from pig liver microsomes could not catalyze the conversion of cobaltic protoporphyrin IX (Co-heme) to biliverdin, although Co-heme could bind with the heme oxygenase protein to form a complex. The heme oxygenase system in the microsomes from pig spleen, rat spleen, and rat kidney also failed to oxidize Co-heme to biliverdin. Properties of the complex of Co-heme and heme oxygenase closely resembled those of cobalt myoglobin and cobalt hemoglobin; the Co-heme bound to the heme oxygenase protein did not react with cyanide and azide, the Co-heme moiety was reduced but only slowly with sodium dithionite, and the reduced form of the Co-heme did not appear to bind carbon monoxide. The co-heme bound to heme oxygenase was not reduced with the NADPH-cytochrome c reductase system in air. These findings further support the views that heme oxygenase may have a heme-binding crevice similar to those of myoglobin and hemoglobin and that reduction of heme is the prerequisite for the oxidative degradation of heme in the heme oxygenase reaction.

Animals↗
PubMed1978

Effects of administration of cobalt chloride and cobalt protoporphyrin on delta-aminolevulinate synthase in rat liver.

Cobalt protoporphyrin inhibited the drug-induced increase of delta-aminolevulinate synthase as well as its transfer from the cytosol fraction to the mitochondria in rat liver in a similar way to protoheme. Cobalt chloride given to animals in a large dose exhibited similar effects. Cobalt protoporphyrin was isolated from the liver of rats treated with cobalt chloride. The observed regulatory effects of cobalt chloride with respect to the induction and the intracellular translocation of delta-aminolevulinate synthase may be mediated by cobalt protoporphyrin synthesized in vivo.

5-Aminolevulinate Synthetase↗
PubMed1977

Reduced level of glycine cleavage system in the liver of hyperglycinemia patients.

The activity of the glycine cleavage system in liver of hyperglycinemia with organic acidemia was 1/6 to 1/20 tha of normal human livers. The reduced activity results from the reduction in concentration of the enzyme of the glycine cleavage system. All the protein components of the glycine cleavage system examined were reduced when the activity was determined seprately. H-protein was purified from patients' and control livers, and there was found no difference in their chromatographic properties.

Carbon Dioxide↗
PubMed1977

Heme oxygenase purified to apparent homogeneity from pig spleen microsomes.

Heme oxygenase was purified to apparent homogeneity from pig spleen microsomes. The purified heme oxygenase showed an apparent molecular weight of 157,000 +/- 7,000 daltons when estimated by gel filtration. On SDS-polyacrylamide gel electrophoresis, the heme oxygenase preparation gave a single protein band showing a minimum molecular weight of about 26,000 daltons. Heme oxygenase could readily bind with heme and the resulting heme complex gave an absorption maximum at 406 nm. The heme bound to the enzyme protein was found to be a good substrate for the heme oxygenase reaction.

Animals↗
PubMed1976

delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.

5-Aminolevulinate Synthetase↗
PubMed1976

Comparative studies of the effects of cyclic AMP, various hormones and chloramphenicol on the induction of delta-aminolevulinic acid synthetase and tyrosine aminotransferase in the organ-cultured chick embryo liver.

Induction of delta-aminolevulinic acid synthetase by allylisopropylacetamide in organ-cultured chick embryo liver was not appreciably influenced by any of cycli AMP, dibutyryl cyclic AMP, theophylline, glucose, insulin, glucagon, epinephrine, isoproterenol, and hydrocortisone, whereas the activity of tyrosine aminotransferase significantly increased in response to cyclic AMP and some of those hormones. Accumulation of delta-aminolevulinic acid synthetase in the cultured liver cytosol fraction was not appreciably increased by the addition of dibutyryl cyclic AMP or insulin to the incubation medium. Apparently the behaviors of the induction of delta-aminolevulinic acid synthetase in chick embryo liver in ovo and in vitro differ from those in the livers of adult chicken and rat. High concentrations of chloramphenicol suppressed significantly the allylisopropylacetamide-induced increase of delta-aminolevulinic acid synthetase as well as incorporation of 14C-leucine into proteins. The activity of tyrosine aminotransferase, however, was rather increased when relatively low concentrations of chloramphenicol were added to the medium.

5-Aminolevulinate Synthetase↗
PubMed1976

Studies on the conversion of the cytosol delta-aminolevulinate synthetase to the mitochondrial enzyme.

DELTA-Aminolevulinate (ALA) synthetase was induced in the rat by allylisopropylacetamide, and ALA synthetase in the liver cytosol fraction was partially purified. The cytosol ALA synthetase shows a molecular weight (M.W.) of about 600,000 by gel filtration and about 178,000 by sucrose density gradient centrifugation, whereas the mitochondrial enzyme gives M.W. about 110,000 by either method. In the presence of 0.25 M NaCl, however, the cytosol ALA synthetase showed M.W. 300,000--350,000 by gel filtration and gave M.W. 110,000 or a S value of 6.4 by centrifugation. Treatment of the cytosol ALA synthetase with papain or snake venom also yielded an entity which showed M.W. 110,000 by gel filtration as well as by sucrose density gradient centrifugation. When the 6.4 S enzyme once separated by centrifugation was combined with a fraction exhibiting about 4 S on centrifugation and was subsequently subjected to gel filtration, the ALA synthetase activity was recovered in the fraction corresponding to M.W. about 170,000. When the combination of the 6.4 S enzyme and a fraction of about 8 S was examined by gel filtration, the ALA synthetase activity was eluted showing M.W. about 250,000. The ALA synthetase in the cytosol fraction seems to exist as a complex composed of an ALA synthetase of M.W. 110,000 and a 4 S protein and an 8 S protein to form a larger size of the enzyme (M.W. 300,000) which further aggregates to give M.W. about 600,000. Physiologically, the complex may be subjected to a limited proteolysis so as to lose the capacity to form complex and aggregates, followed by incorporation of smaller sizes of the enzyme into the mitochondria.

5-Aminolevulinate Synthetase↗