Rat Liver Ornithine Decarboxylase Research Articles

Effects of hydrazine and its derivatives on ornithine decarboxylase synthesis, activity, and inactivation

Hydrazine (1.0 mmol/kg) caused marked stimulation of rat liver ornithine decarboxylase (ODC) activity. Derivatives of hydrazine were less effective as stimulators. Enzyme activity reached a maximum about 4 hr after treatment. ODC activity in livers from dexamethasone stimulated rats was moderately inhibited when hydrazine (1.0 mmol/kg) was administered 20 min prior to sacrifice. Hydrazine also slowed inactivation halftime of ODC following dexamethasone stimulation, as measured by serial biopsy in single animals. The inactivation half-times of the treated animals fell into two populations, one responding more extensively than the other. The usefulness of a serial liver biopsy technique for determination of ODC half-time in individual animals is demonstrated.

The effect of vasopressin on the activity of ornithine decarboxylase in rat brain and liver.

The effect of vasopressin on the activity of ornithine decarboxylase in rat brain and liver.

Diamine-induced inhibition of liver ornithine decarboxylase

Repeated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ;antienzymes' [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858-1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme-inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

Effects of dietary butylated hydroxytoluene and phenobarbital on the activities of ornithine decarboxylase and thymidine kinase in rat liver and lung

Rats were entrained to a 12 h dark/12 h light cycle with food (60% protein +/- 0.5% butylated hydroxytoluene or 0.05% phenobarbital) available only during the first 2 h of the dark period. Under these conditions liver ornithine decarboxylase (ODC) activity in control animals displayed a characteristic diurnal oscillation. In livers of rats fed butylated hydroxytoluene or phenobarbital ODC activity was not increased whereas thymidine kinase (TK) activity was stimulated 4--10 fold at 3 days. In lungs from the same animals ODC and TK activities were unchanged. In rats fed butylated hydroxytoluene for 3 days [3H]thymidine incorporation into DNA was increased in liver but decreased in lung.

Stabilization of ornithine decarboxylase in rat liver

The response of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity to cyloheximide has been measured in rat liver after various stimuli known to enhance the enzyme activity. While a single injection of cycloheximide almost totally (> 95%) inhibited the activity of ornithine decarboxylase in livers of growth-hormone treated rats at 45 min after the administration of the drug, this inhibition was much less striking in livers of partially hepatectomized (80%), of thioacetamide-treated (60%) and especially of carbon-tetrachloride treated (15%) animals thus suggesting a stabilization of the enzyme after the latter treatments. The measurement of the apparent half-life of ornithine decarboxylase after cycloheximide revealed a substantial decrease in the decay rate of the enzyme in livers of thioacetamide-treated rats. The apparent half-life of S- adenylsyl- l - methionine decarboxylase (EC 4.1.1.50) likewise appeared to be longer in rats previously treated with thioacetamide or carbon tetrachloride than in partially hepatctomized animals. Injection of thioacetamide also resulted in an initial stabilization of liver ornithine decarboxylase against 1,3-diaminopropane, a potent indirect inhibitor of the enzyme. However, 30 min after diaminopropane injection the enzyme activity started to fall extremely rapidly. This sudden drop in ornithine decarboxylase activity following diaminopropane could be largely prevented with cycloheximide. This finding clearly indicates that a protein-synthesis dependent formation of macromolecular inhibitors of ornithine decarboxylase may also be an early event after amine treatments. The striking stabilization of ornithine decarboxylase activity following these treatments, which are known to produce an initial liver necrosis (especially carbon tetrachloride and thioacetamide), may indicate that the system(s) responsible for the rapid inactivation of liver ornithine decarboxylase is more sensitive to tissue necrosis than the enzyme itself.

Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed.

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...

Detection of multiple forms of rat liver ornithine decarboxylase.

Rat liver ornithine decarboxylase induced by injection of thioacetamide has been separated into at least two fractions by covalent chromatography on an activated thiol-Sepharose 4B column. The two major fractions could be distinguished by ion exchange chromatography and electrophoresis on acrylamide gels. In addition, the two forms displayed different Km values for ornithine. Although the two forms are separable, they display identical antigenic properties, pH optima, and they appear to be the same molecular size. The biological significance or the relationship between multiple forms of ornithine decarboxylase is not understood.

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.

Changes in rat liver ornithine decarboxylase activity during ageing and effect of stimulation by dexamethasone

We have studied the ornithine decarboxylase activity in the liver of 1, 3 and 24 months old rats: such activity has a dramatic diminuation in the age range from 1 to 3 months, but the activity at 24 months is significantly higher than that at 3 months. The treatment with Dexamethasone causes a very remarkable elevation in the activity of ornithine decarboxylase, up to rates that are not significantly different for the three ages.

Stable multisubstrate adducts as enzyme inhibitors: Potent inhibition of ornithine decarboxylase by N-(5′-phosphoyridoxyl)-ornithine

The synthesis of several N-(5′-phosphopyridoxyl)-amino acids is described. These compounds, analogs of the Schiff base intermediate involved in enzyme-catalyzed decarboxylation, are potent inhibitors of the cognate amino acid decarboxylases. Kinetic studies using partially purified rat liver ornithine decarboxylase, have shown that N-(5′-phosphopyridoxyl)-ornithine inhibits the enzyme in a non-competitive manner with respect to both ornithine and pyridoxal-5′-phosphate. These findings suggest that the inhibitor binds to the holoenzyme active site in plact of the Schiff base intermediate.

Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.

Relation between growth hormone and thioamide derivatives in induction of ornithine decarboxylase in rat liver.

Relation between growth hormone and thioamide derivatives in induction of ornithine decarboxylase in rat liver.

Stimulation of ornithine decarboxylase and nuclear RNA polymerase activity in rat liver by glucagon and dibutyryl cyclic amp.

ABSTRACT The influence of glucagon and dibutyryl cyclic 3′,5′-AMP on ornithine decarboxylase (EC 4.1.1.17) and nuclear RNA polymerase (EC 2.7.7.6) activity of rat liver was studied. Intraperitoneal administration of 0.2 mg of glucagon per 100 g body weight resulted in a four- to six-fold increase in ornithine decarboxylase activity. This enzyme activity was also markedly increased by a single ip injection containing 2.5–5 mg of dibutyryl cyclic AMP. The time course of this effect demonstrated a maximum at 3–4 h. Both agents also stimulated nuclear RNA polymerase activity. The increase in hepatic ornithine decarboxylase activity by dibutyryl cyclic AMP was not abolished by adrenalectomy but was totally prevented by the administration of actinomycin D, α-amanitin and cycloheximide, which suggests that the increase in activity represents synthesis de novo of the enzyme. A sharp rise in liver ornithine decarboxylase activity was also observed after administration of theophylline, whereas no change in enzyme activity was found after the administration of adenosine or adenosine 5′-nucleotides.

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

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.

Puromycin Stimulation of Rat Liver Ornithine Decarboxylase Activity

IT is well known that puromycin inhibits protein synthesis in mammalian systems1, but other metabolic effects have been reported2–5. Our own work on the purification of rat liver ornithine decarboxylase (ODC)6, the apparent rate-limiting enzyme in polyamine synthesis, led us to a study of hormonal factors which alter the activity of this enzyme. We found that dibutyryl-cyclic adenosine 3′,5′-monophosphate (dibutyryl-cyclic AMP) stimulated hepatic ODC activity in normal rats6a. When actinomycin D, which had no independent effect on the basal level of the enzyme, was given with dibutyryl-cyclic AMP, ODC activity was inhibited. However, when the animals were dosed with either puromycin alone or puromycin and dibutyryl-cyclic AMP at levels of puromycin which inhibited protein synthesis, the antibiotic alone stimulated ODC activity and potentiated the activity due to dibutyryl-cyclic AMP.

The [formula omitted] stimulation of rat liver ornithine decarboxylase activity by dibutyryl cyclic adenosine 3′,5′-monophosphate, theophylline and dexamethasone

The hepatic ornithine decarboxylase (ODC) activity of normal rats was stimulated more than 7-fold 3 hours after a single intraperitoneal injection of dibutyryl cyclic adenosine 3′,5′-monophosphate (dibu-cAMP). The 3-hour ODC activity was also stimulated by single injections of either theophylline or dexamethasone (10- and 21-fold, respectively). The simultaneous administration of actinomycin D with either dibu-cAMP, theophylline or dexamethasone reduced the 3-hour ODC activity by 91, 62 and 58 percent, respectively. When actinomycin D was given one hour after dibu-cAMP, no inhibition of ODC activity was observed.

The control of ornithine decarboxylase activity during liver regeneration

The effect of amino acids and growth hormone on rat liver ornithine decarboxylase activity was investigated in an attempt to elucidate the mechanisms that control the activity of this enzyme during the early stages of liver regeneration. A single administration of casein hydrolyzate to intact rats produces a marked increase in liver ornithine decarboxylase activity. The casein effect is abolished by puromycin or actinomycin, is not modified by adrenalectomy and is slowed down by hypophysectomy. Histidine also causes a rapid increase in the activity of the enzyme but the amino acid effect is blocked by puromycin, is not inhibited by actinomycin, and is entirely absent in hypophysectomized rats. Growth hormone injections increase ornithine decarboxylase activity in normal or regenerating liver. The action of the hormone is only slightly inhibited by doses of actinomycin that prevent any increase in the specific activity of nuclear or cytoplasmic rapidly labeled RNA. 8-Azaguanine injection or small doses of actinomycin do not block the initial increase in ornithine decarboxylase activity occurring shortly after partial hepatectomy. The possibility that a post-transcriptional mechanism is involved in the control of ornithine decarboxylase activity during liver regeneration is discussed in detail.

Growth hormone induction of ornithine decarboxylase in rat liver.

The effect of growth hormone on hepatic ornithine decarboxylase (L-ornithine carboxy- lyase, EC 4.1.1.17) (ORD) activity was studied in adult and weanling rats. Single injections of growth hormone produced up to 30-fold increases in ORD activity in both adults and weanlings. However, with lower doses of growth hormone, a greater increase in ORD activity occurred in weanlings than in adults. Both adult and weanling rats showed the same time course of ORD activity enhancement after growth hormone with peak enzymatic activity at 4 hr and a decline to basal levels by 8 hr after hormone administration. In weanling rats, cycloheximide completely prevented the elevation of ORD activity at 4 hr after growth hormone if administered any time up to 3 hr after the hormone. If given simultaneously with growth hormone, actinomycin D almost totally supressed the enhanced ORD activity 4 hr after growth hormone. However, the ability of actinomycin D to prevent increased ORD activity decreased as the time after growth horm...