Yoshiro Takeda

Studies on ornithine decarboxylase from the liver of thioacetamide-treated rats. Purification and some properties.

Abstract Ornithine decarboxylase ( l -ornithine carboxy-lyase, EC 4.1.1.17) was purified about 5400-fold from the soluble fraction of liver from thioacetamide-treated rats. The purified enzyme showed only a single protein band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be approx. 100 000. Its isoelectric point was found to be at pH 4.1 and its optimal pH around 7.0. The Km value for l -ornithine was 2·10−4 M at pH 7.0. The enzyme required thiol compounds for maximal activity and was inhibited by inhibitors of pyridoxal enzymes, such as l -canaline and isonicotinic acid hydrazide. Among the various amino acids and amines tested, putrescine and d -ornithine caused a weak inhibition, while other compounds had little effect. The properties of ornithine decarboxylase from the liver of thioacetamide-treated rats were compared with those of enzyme from regenerating rat liver.

Dietary response of various key enzymes related to glucose metabolism in normal and diabetic rat liver

Abstract With normal rats, administration of a high glycerol diet for 3 days produces a great increase in the activities of various key enzymes involved in glucose utilization in the liver. These include glucokinase (EC 2.7.1.12), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), ATP citrate lyase (EC 4.1.3.8) and acetyl-CoA carboxylase (EC 6.4.1.2). Administration of a high glycerol diet to diabetic animals also causes a marked induction of all these enzymes, except glucokinase and glucose-6-phosphate dehydrogenase, in the liver. The latter two enzymes show little response to glycerol feeding in the diabetic state. The induction of pyruvate kinase nd ATP citrate lyase by glycerol feeding is almost completely abolished by actinomycin D treatment in both normal and diabetic rats. The elevated activities of glucose-6-phosphatase (EC 3.1.3.9) and l -serine dehydratase (EC 4.2.1.13) in diabetic liver are not lowered but rather increase on feeding glycerol. A possible explanation for these results is presented, especially in relation to the action of insulin.

Metabolism of p-hydroxyphenylacetic acid in pseudomonas ovalis

Abstract From whole-cell and cell-free experiments, it was found that 3,4-dihydroxyphenylacetic acid was an intermediate in p -hydroxyphenylacetate oxidation in Pseudomonas ovalis . p -Hydroxyphenylacetate hydroxylase and 3,4-dihydroxyphenylacetate oxygenase were extracted from cells grown on p -hydroxyphenylacetic acid and were partially purified. The former enzyme was separated into two protein components, and in its reaction 1 mole of oxygen was consumed per mole of substrate in the presence of NADH 2 . The latter enzyme catalyzed the ring fission reaction between C-2 and C-3 of the benzene nucleus of 3,4-dihydroxyphenylacetic acid, consuming 1 mole of oxygen per mole of substrate. The product of this ring fission reaction was identified as δ-carboxymethyl-α-hydroxymuconic semialdehyde.

Changes in polyamine metabolism during wound healing in rat skin

Abstract Changes in the activity of ornithine decarboxylase ( l -ornithine carboxylyase, EC 4.1.1.17) during the early stages of wound healing in rat skin were investigated in comparison with those of histidine decarboxylase ( l -histidine carboxy-lyase, EC 4.1.1.22). Ornithine decarboxylase activity increased very rapidly after wounding and reached a maximum after 12 h. Then it decreased rapidly and returned to normal after 48 h. Histidine decarboxylase activity increased more slowly reaching a maximum after about 24 h. The elevation of ornithine decarboxylase activity was followed by increase in the concentration of polyamines in the wounded skin. Administration of betamethasone, a potent synthetic glucocorticoid, delayed the rise in ornithine decarboxylase activity in the wounded tissues, but not the rise in histidine decarboxylase activity.

Purification and properties of homogentisate oxygenase from Pseudomonas fluorescens

Summary Homogentisate oxygenase from Pseudomonas ftuorescens adapted to tyrosine has been isolated, purified and crystallized. The crystallized enzyme was homogeneous on ultracentrifugation, and its sedimentation constant was found to be 11.8 S. The molecular weight of the enzyme was calculated to be about 380 000. Like the mammalian liver enzyme, bacterial homogentisate oxygenase requires ferrous iron as a cofactor. For maximal activity, the enzyme requires at least 10 min preincubation with ferrous iron at pH 6.0. The optimal pH is 6.0. Both glutathione and ascorbate are also required for maximal activity at pH 6.0, but only the former is essential at pH 5.4. The Km values are 6 · 10−4 M for homogentisate and 1 · 10−4 M for Fe2+ at pH 6.0. Unlike the mammalian liver enzyme, the bacterial enzyme is fairly stable on aging or storage. p-Chloromercuribenzoate inhibits the enzyme with respect to ferrous iron, but non-competitively with respect to homogentisate. The properties of bacterial homogentisate oxygenase are discussed in comparison with those of the mammalian liver enzyme.

The role of ATP citrate lyase in the transfer of acetyl groups in rat liver

The role of ATP citrate lyase (ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating), EC 4.1.3.8) in the transfer of acetyl groups out of the mitochondria was investigated in rat liver its anti-enzyme. n nWith a reconstructed system from normal rat liver (fed state), containing both the supernatant and the mitochondria, treatment of the supernatant with the antiserum reduced the acetylation of either sulfanilamide or p-toluidine by pyruvate to about 15% of the obtained by treatment of the supernatant with control serum. These drops in the rate of acetylation were completely reversed by addition of excess purified ATP citrate lyase. With mitochondria alone, it was found that the acetylation of these compounds was parallel to the amount of ATP citrate lyase added. Similar degrees of inhibition of the acetylation of p-toluidine by the antibody were obtained with preparations from the livers of fasted or diabetic rats. In addition, neither (−)- nor (+)-carnitine affected the acetylation rate under any conditions. From these findings, it was concluded that more than 80% of the extramitochondrical acetyl-CoA, derived from pyruvate via the mitochondria, is supplied through the ATP citrate lyase pathway in rat liver, even in the fasted or diabetic state.

The role of putrescine in cell proliferation of the skin of mice induced by ethylphenylpropiolate

Abstract The activities of ornithine and S-adenosylmethionine decarboxylases in the skin of the back of mice increased rapidly after a single topical application of ethylphenylpropiolate (EPP), a potent hyperplastic agent of epidermis, the increase in the former being much greater and more rapid. The tissue concentration of putrescine changed in parallel with change in ornithine decarboxylase activity and was maximal 8 hr after EPP treatment. The spermidine level descreased significantly 4–6 hr after EPP treatment, when putrescine formation from spermidine was accelerated, and increased to 20% more than the normal level at 20 hr. The spermine level did not change within 28 hr after EPP treatment. Administration of dl -α-hydrazino-δ-aminovaleric acid ( dl -HAVA), an inhibitor of ornithine decarboxylase, greatly inhibited the increase in putrescine but had little effect on the change in spermidine concentration after EPP treatment. Examination of DNA synthesis and the histological appearance of the skin showed that dl -HAVA also inhibited inducton of cell proliferation by EPP. The inhibition by dl -HAVA was reversed by administration of putrescine, but not cadaverine or 1,7-diaminoheptane. From these results, it is suggested that the rise in the putrescine level induced by EPP is a requisite for subsequent cell proliferation in the skin of mice.

Effect of thioamide derivatives on induction of ornithine decarboxylase in rat liver

Abstract The effects of various thioamide derivatives on induction of ornithine decarboxylase ( L -ornithine carboxy-lyase, EC4.1.1.17) in rat liver were investigated. Among the thioamides tested, thioacetamide and acylthioureas, having less than 10 carbon atoms in the acyl group, were all effective in increasing enzyme activity in both normal and adrenalectomized rats. Induction of ornithine decarboxylase by thioacetamide was not affected by hypophysectomy, while that by acylthioureas, including hexanoylthiourea, was much less in the absence of the pituitary. When given together with growth hormone, hexanoylthiourea greatly amplified the action of growth hormone in increasing ornithine decarboxylase activity in hypophysectomized rats. This amplifying effect of hexanoylthiourea was also observed in normal rats. The elevation of ornithine decarboxylase activity by hexanoylthiourea was followed by increase in the levels of putrescine and spermidine in the liver.

RESPIRATORY CONTROL OF DISPERSED RAT-LIVER CELLS.

Abstract The respiration of dispersed rat-liver cells was measured using an oxygen electrode and conventional manometric techniques. It was found that cellular respiration was regulated by oxidative phosphorylation. This was shown by the marked stimulation of cellular respiration caused by addition of ADP. However, a considerable difference was found in the regulatory patterns of cellular and mitochondrial respiration. At the cellular level, the transition from State 3 to State 4 was gradual, whereas at the mitochondrial level it was abrupt. In addition, cell suspensions maintained their respiratory control in a medium free of polyols. ATP formation in disperesed cells was shown by incorporation of inorganic [ 32 P]phosphate and consumption of added inorganic phosphate.

PROTEIN AND RIBONUCLEIC ACID SYNTHESES IN DISPERSED RAT-LIVER CELLS.

Abstract Using dispersed rat-liver cells, protein and RNA syntheses were studied. Dispersed cells did not show any obligatory requirements for protein synthesis. Among the various media tested, 10 % polyvinylpyrrolidone and Lockes solution were the most suitable for protein synthesis, while 0.1 M sucrose was the best for lipid synthesis. The rates of protein and RNA syntheses in dispersed cells proceeded linearly during the first 2 h of incubation, whereas those of a homogenate decreased with time reaching a steady level after 30 min. The incorporation of amino acid into protein was inhibited by 2,4-dinitrophenol and anaerobiosis. Puromycin also strongly inhibited amino acid incorporation into protein but not RNA synthesis. Actinomycin strongly inhibited the incorporation of uridine into RNA, but, when not preincubated with the cells, it was less inhibitory for protein synthesis. However, when it was preincubated with the cells, the degree of inhibition of amino acid incorporation into protein by this antibiotic increased and reached a maximal value when the incubation period was 1 h. These results support the idea that protein synthesis in dispersed cells is controlled by the formation of messenger RNA.