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Biomedical subjects

G Kikuchi

Publications and source records attributed to G Kikuchi.

At least 55 records · Page 3Linked to original sources
PubMed1981

Photo-reversal by monochromatic light of the carbon monoxide-inhibited heme degradation catalyzed by the reconstituted heme oxygenase system.

Photo-reversal of the carbon monoxide inhibition of heme oxygenase reaction by monochromatic light was investigated. Heme degradation in either the microsomal or the reconstituted heme oxygenase system was inhibited by CO. In both systems the extents of Co inhibition were dependent on the CO/O2 ratio and were nearly equal at a given CO/O2 ratio. In the reconstituted heme oxygenase reaction using a highly purified heme oxygenase preparation the relationship between the intensity of light and the degree of reversal of the CO inhibition of heme degradation as expressed in terms of delta K/Kd was not linear, but the tentatively obtained photochemical action spectrum exhibited the peaks of reversal at about 420, 540, 570, and 640 nm and suggested the occurrence of at least two steps of CO inhibition in the overall sequence of heme degradation. One could be ascribed to protoheme and the other was supposed to be the 688 nm compound which is an intermediate locating between hydroxyheme and the biliverdin-iron complex in the sequence of heme degradation.

Animals↗
PubMed1981

Defective glycine cleavage system in nonketotic hyperglycinemia. Occurrence of a less active glycine decarboxylase and an abnormal aminomethyl carrier protein.

The activities of then glycine cleavage system in the liver and brain of patient with nonketotic hyperglycinemia was extremely low as compared with those of control human liver and brain. The activities of glycine decarboxylase (P-protein) and the aminomethyl carrier protein (H-protein), two of the four protein components of the glycine cleavage system, were considerably reduced in both the liver and brain; the extent of reduction was greater in the H-protein. The activity of the T-protein was normal. Purified H-protein from the patient did not react with lipoamide dehydrogenase, and titration of thiol groups with [2,3-14C]N-ethylmaleimide suggested that this H-protein is devoid of lipoic acid. This structural abnormality in the H-protein is considered to constitute the primary molecular lesion in this patient with non-ketotic hyperglycinemia. Immunochemical studies using an antibody specific for P-protein showed that the patient was due to reduction of the catalytic activity of the protein rather than a decrease in the actual amount of the P-protein. Partial inactivation of P-protein could result secondarily from impaired metabolism of glycine resulting from deficiency in the activity of H-protein. However, the H-protein from the patient could stimulate the P-protein catalyzed exchange of the carboxyl carbon of glycine with 14CO2, although the specific activity of the purified H-protein from the patient was only 4% of that of control human H-protein. The content of H-protein in the liver of the patient was approximately 35% of that of control human liver.

Amino Acid Metabolism, Inborn Errors↗
PubMed1980

The mitochondrial glycine cleavage system. Purification and properties of glycine decarboxylase from chicken liver mitochondria.

Glycine decarboxylase, tentatively called P-protein and considered a constituent of the glycine cleavage system, was purified to apparent homogeneity from chicken liver mitochondria. P-protein is a homodimer having a Mr = approximately 200,000 and consisting of identical subunits with Mr = approximately 100,000. Each subunit appears to contain an equimolar pyridoxal 5'-phosphate which is bound to the protein, possibly through a protonated aldimine linkage. The isoelectric point of P-protein was 7.2. P-protein could bind glycine, showing a Kd of 33 mM for it, and could catalyze glycine decarboxylation even though the rate of decarboxylation catalyzed by P-protein alone was extremely low. The product of glycine decarboxylation was methylamine and the Km for glycine. Methylamine could bind to P-protein, giving a Kd value of 63 mM, and it inhibited the glycine decarboxylation. P-protein alone could also slightly catalyze the exchange of carboxyl carbon of glycine with CO2 and the exchange appeared to obey a ping-pong mechanism. Both glycine decarboxylation and the glycine-CO2 exchange catalyzed by P-protein were stimulated 100-fold or more by the addition of lipoic acid, which is a functional group of H-protein. We may define P-protein as glycine decarboxylase although P-protein alone exhibits only very low catalytic activities.

Amino Acid Oxidoreductases↗
PubMed1980

On the nature of three forms of phosphoenolpyruvate carboxykinase occurring in the cytosol of chicken liver.

Chicken liver mitochondria contained two forms of phosphoenolpyruvate (PEP) carboxykinase (designated as Mt-I and Mt-II) and the chicken liver cytosol fraction contained three forms of PEP carboxykinase (designated as Sol-I, Sol-II, and Sol-III). Mt-I and Mt-II were purified to homogeneity. They both had a molecular weight of 72,000 as judged by the sodium dodecyl-sulfate-polyacrylamide gel electrophoresis method, whereas an apparent molecular weight of 42,000 was obtained for both enzymes when estimated by Sephadex G-100 gel filtration. Studies with a rabbit antibody against the purified Mt-I revealed that Mt-I, Mt-II, Sol-I, and Sol-II are immunochemically identical, whereas Sol-III is immunochemically different from any of the other proteins. Genetic analogy between Mt-I and Mt-II was also suggested by gel electrophoretic analysis of their peptide maps. The content of Sol-III in the adult chicken liver was very small, whereas the livers of chick embryo and very young chick contained a considerable amount of Sol-III. The level of Sol-III in the adult chicken, however, could be significantly increased by the administration of hydrocortisone or isoproterenol. Apparently Sol-III is a cytosol-specific PEP carboxykinase which is similar to the cytosolic PEP carboxykinases of various mammals. Sol-III showed a molecular weight of about 60,000 as estimated by gel filtration. Studies with combined use of the antibody and [3H]leucine revealed that some portion of PEP carboxykinase (Sol-I plus Sol-II) appearing in the liver cytosol corresponds to a precursor in transit to the mitochondria, although major portions of Sol-I and Sol-II appear to be accounted for by release of the mitochondrial enzymes.

Animals↗
PubMed1980

Purification and properties of glycine decarboxylase, a component of the glycine cleavage system, from rat liver mitochondria and immunochemical comparison of this enzyme from various sources.

Glycine decarboxylase, tentatively called P-protein as a constituent of the glycine cleavage system, was purified to near homogeneity from rat liver mitochondria. The purified P-protein was a homodimer with a molecular weight of about 210,000, consisting of identical subunits with a molecular weight of 105,000. In the exchange reaction of the carboxyl carbon of glycine wih CO2 catalyzed by the purified P-protein in the presence of H-protein, the pH optimum was 6.7, Km for glycine was 6.6 mM, and Km for H-protein was 7.4 microM. A specific rabbit antibody against the purified rat liver P-protein was prepared. Ouchterlony double diffusion analysis and immunoinhibition experiments using this antibody revealed immunological cross-reactivity among the P-proteins from various species of animals such as carp, frog, snake, chicken, bovine, and human, suggesting a quite conservative evolution of the glycine cleavage system.

Amino Acid Oxidoreductases↗
PubMed1980

Immunochemical studies of the turnover of delta-aminolevulinate synthase in rat liver mitochondria and the effect of hemin on it.

The half-life of delta-aminolevulinate (ALA) synthase in the liver mitochondria of allylisopropylacetamide-treated rats was estimated to be about 35 min, taking advantage of the observation that hemin strongly inhibits the translocation of ALA synthase from the liver cytosol into the mitochondria. ALA synthase accumulating in the liver cytosol in allylisopropylacetamide-treated rats is rapidly translocated into mitochondria with a half-disappearance time of about 20 min, but does not appear to undergo degradation until it is translocated into mitochondria. A study with an ALA synthase-specific IgG revealed that the decrease in the ALA synthase activity in the liver mitochondria observed after the administration of cycloheximide or hemin effectively paralleled the decrease in the amount of immunologically reactive protein, providing further evidence that ALA synthase is rapidly degraded in the mitochondria.

5-Aminolevulinate Synthetase↗
PubMed1980

Effects of hemin on the synthesis and intracellular translocation of delta-aminolevulinate synthase in the liver of rats treated with 3,5-dicarbethoxy-1,4-dihydrocollidine.

The administration of hemin to rats which had been treated with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) brought about a rapid decrease of the delta-aminolevulinate (ALA) synthase activity in the mitochondria and an appreciable increase of the enzyme activity in the cytosol in the liver. In DDC-treated rats, however, the degree of accumulation of ALA synthase in the liver cytosol resulting from the hemin administration was relatively small and the total ALA synthase activity in the liver was considerably decreased after the hemin administration; these results are in contrast with those for allylisopropylacetamide-treated rats. Further studies by combined use of [3H]leucine and an anti-ALA synthase IgG revealed that incorporation of [3H]leucine into the mitochondrial ALA synthase was greatly reduced in hemin-injected rats and that incorporation of [3H]leucine into total ALA synthase in the liver (the sum of radioactivity incorporated into the mitochondrial enzyme and the cytosolic enzyme) was also decreased to a considerable extent in these rats; this decrease in incorporation of [3H]leucine appeared to occur immediately after the administration of hemin. It was concluded that hemin actually inhibits the translocation of ALA synthase from the cytosol into the mitochondria and that hemin also inhibits de novo synthesis of ALA synthase, possibly at a post-transcriptional level in DDC-treated rats.

5-Aminolevulinate Synthetase↗
PubMed1980

Intracellular site of synthesis of microsomal heme oxygenase in pig spleen.

In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.

Animals↗