Roichi Itoh

5'-nucleotidase of chicken liver.

1. 1.|5′-Nucleotidase (5′-ribonucleotide phosphophydrolase, EC 3.1.3.5) was partially purified from chicken liver. This is the first time it has been possible to obtain 5′-nucleotidase from the hepatic tissue of uricotelic animals and it was found to be kinetically distinct from 5′-nucleotidases obtained from other sources. 2. 2.|5′-Mononucleotides having a keto group at position 6 in the purine base are the most active substrates of this enzyme. 5′-IMP is the most active substrate among the 5′-nucleotides tested, and it is about 10 times more active than 5′-AMP. 3. 3.|This enzyme has an optimum pH at 6.5 and requires divalent metal ions. In the absence of divalent metal ions, the enzyme is almost inactive. 4. 4.|Inosino, guanosine p-chloromercuribenzoate (PCMB) and NaF inhibit this enzyme. Of these inhibitors, PCMB was found to be the most potent. 5. 5.|The general properties of the enzyme are described, and its possible metabolic function is discussed.

Kinetic properties of cytosol 5'-nucleotidase from chicken liver.

Abstract A highly purified preparation of cytosol 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) from chicken liver was effectively activated by ATP and inhibited by P i . When AMP was used as substrate, the enzyme displayed sigmoidal kinetics. When either IMP, GMP, UMP or CMP was used as substrate, the substrate saturation curve was slightly sigmoidal or hyperbolic. In the presence of 10 mM ATP, the substrate saturation curves for all substrates tested were hyperbolic, whereas P i increased the sigmoidicity of the saturation curves. The enzyme had much higher affinities for IMP and GMP than for AMP, UMP and CMP. ATP decreased the s 0.5 , whereas P i increased the s 0.5 for all the substrates tested. Inhibition by P i was removed by ATP, and activation by ATP was removed by P i . V of the enzyme was determined to be in the same order of the magnitude for all the substrates tested. The hydrolysis of GMP was inhibited competitively by the other 5′-nucleotides. AMP at low concentration had a stimulatory effect on the hydrolysis of GMP. The enzyme activity was absolutely dependent on the presence of bivalent cations. Increasing concentration of MgCl 2 increased the V and affinity of the enzyme for AMP and vice versa. ATP protected the enzyme against the inactivation by heat or trypsin digestion. It seems likely that this enzyme is an allosteric protein regulated by various ligands through conformational changes.

Changes in 5′-nucleotidase activity in chick liver during development and dietary treatment

Abstract 1. 1. To elucidate the physiological roles of hepatic 5′-nucleotidase (EC 3. I .3.5), changes in IMP-hydrolysing activity in chick liver extracts during dietary adaptation were investigated. 2. 2. IMP-hydrolysing activity in chick liver extracts increased 2-fold within one day when the casein content of the diet was increased from 5 to 75%. 3. 3. Increased IMP-hydrolysing activity was Mg 2+ -dependent and has a pH optimum at about 6.5. 4. 4. A correlation between IMP-hydrolysing activity in chick liver extracts and serum uric acid concentration was observed. 5. 5. In rat liver extracts, no dependency of IMP-hydrolysing activity upon the casein content of the diet was observed. 6. 6. Within two days after hatching, a 2-fold increase in IMP-hydrolysing acitivity in chick liver extracts was observed compared with the activity immediately before hatching. 7. 7. These results suggest that the 5′-nucleotidase, which has been described by us previously 1 , would play a role in the excretory metabolism of α-amino nitrogen of amino acid in a uricotelic animal.

5′-Nucleotidase of chicken liver: A comparison of soluble 5′-nucleotidase activities in chicken and rat liver

Abstract 1. 1. Most of the IMP-hydrolyzing activity in the homogenate of chicken liver was recovered in the cell sap. This activity, which has a pH optimum of around 6-5 and is Mg 2+ dependent, is due to the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) previously described (Itoh, Mitsui & Tsushima, 1967). 2. 2. The activity of this enzyme in chicken liver was much higher than in rat liver.

Involvement of DNA polymerase δ and/or ϵ in joining UV-induced DNA single strand breaks in human fibroblasts (comparison of effects of butylphenyldeoxyguanosine with aphidicolin)

DNA polymerases involved in ultraviolet (UV)-induced DNA repair were studied in human fibroblasts using the inhibitors of DNA polymerases, aphidicolin which inhibits DNA polymerases alpha, delta and epsilon, and butylphenyldeoxyguanosine (BuPGdR) which inhibits DNA polymerase alpha strongly and weakly inhibits delta and epsilon. Both inhibitors inhibited replicative DNA synthesis in a dose dependent manner as measured by thymidine incorporation. However, BuPGdR did not accumulate single strand breaks in cells irradiated with 5 J/m2 UV-light even at the highest dosage tested, indicating that BuPGdR does not inhibit DNA repair. On the other hand, aphidicolin accumulated single strand breaks in UV-light irradiated cells. These results suggest that DNA polymerase delta and/or epsilon are mainly involved in UV-induced DNA repair.

Sequence homology of Chinese hamster metallothionein genes I and II to those of the mouse and rat, and their amplification in Cd-resistant cells

The metallothionein (MT) I and II genes were isolated from Chinese hamster cells and sequenced. The MT-II gene is located about 6 kb upstream of the MT-I gene and their arrangement is similar to those of the mouse and rat MT genes. The sequence of the Chinese hamster MT-I gene is highly homologous to those of the mouse and rat, particularly in their promoter regions of MT-I. However, the promoter region of MT-II has less homology with those of the mouse and rat due t to insertions and deletions. The MT-I and MT-II genes were equally amplified 4-8-times in the Cd-resistant Chinese hamster cells, suggesting that both genes are included in the same amplification unit. Cytogenetic analysis of Cd-resistant cells by in situ hybridization showed that they are randomly integrated into multiple sites on the chromosomes.

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties

1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.

Chicken liver amidophosphoribosyltransferas Ligand-induced alterations in molecular properties

A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.

A comparative study of hypoxanthine phosphoribosyltransferase activity in birds and mammals

Abstract 1. 1. Hypoxanthine phosphoribosyltransferase activities in birds were compared with those in mammals. 2. 2. In the livers of chicken, quail and pigeon, the activities were much lower than that in rat liver, though the activities in the avian kidney were on almost the same level as that of rat kidney. 3. 3. The incorporation of [14C]hypoxanthine into acid soluble nucleotides and RNA was also very low in the avian liver.

A comparative study of formiminotransfer from formiminoglutamic acid to tetrahydrofolic acid in animals

Abstract 1. 1. Formiminotransfer activity from formiminoglutamic acid to tetrahydrofolic acid was detected in hepatic tissue of bird, reptile, amphibian, fish, a crustacean and a mollusc. 2. 2. Several properties of transfer activity partially purified from avian liver were apparently the same as those of mammalian enzyme.