Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Studies on mechanisms of ornithine decarboxylase activity regulation in regenerating liver

Rat liver (hydrocortisone-induced) ornithine decarboxylase has been shown to be stable when the cytosolic fraction is incubated alone at 37 degrees C, although there is a very rapid and drastic loss of activity after addition of microsomes to the incubation medium. The present paper is concerned with the behaviour of ornithine decarboxylase induced in rat liver by a growth stimulus (partial hepatectomy); comparative studies have been carried out on the enzyme induced by sham operation, or by hydrocortisone. Results show that ornithine decarboxylase from regenerating liver is more stable when incubated with microsomes (from the same source); this higher stability depends both on a lower microsome-bound inactivating capacity and a limited susceptibility of the enzyme to the inactivation. A critical role in modulating the microsome-dependent inactivation appears to be played by low molecular weight cytosolic factors, whose greater content in regenerating liver is likely to be included with the factors above in determining the relative stability of ornithine decarboxylase.     

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

Zinc can influence ornithine decarboxylase activity in rat thymus cells

Summary The thymus of young rats contained a high basal activity of ornithine decarboxylase (ODC). Treatment with zinc sulphate caused a slight increase of thymic ODC activity within 6 hours and a more marked enhancement (three-fold) in the spleen 24 h after treatment. In spite of the high activity of thymic ODCin vivo, ODC was not detectable in primary cultures of rat thymocytes, but was early and largely induced after treatment with Concanavalin A (Con A). The presence of 0.1 mM zinc in the medium increased the response of ODC to Con A. This effect of zinc in mitogen activated thymocytes may be due to the stabilization of ODC, which was found to decay with a half life of 65 min after the block of protein synthesis with cycloheximide. On the contrary in absence of zinc the half life of the enzyme was 40 min, as in the rat thymus in vivo.Zinc alone, at 0.1 mM concentration, did not affect ODC activity in resting thymocytes during the early times, but the metal was able to cause an increase of the enzyme activity after 4–6 days of culture. Other heavy metals such as mercury, cadmium and copper provoked a late increase of ODC activity, but their action was evident only at dosages which were toxic for the cells.     

Effect of sodium arsenite on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 microM arsenite and complete inhibition was obtained at concentrations above 10 microM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half-life of ODC activity, measured after cycloheximide treatment, increased almost six-fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol-redox status in cells, also caused a similar effect on the induction and stability of ODC.     

Rapid loss of ornithine decarboxylase activity during encystation of Entamoeba invadens

Abstract Stimulation of encystation of Entamoeba invadens by incubation of trophozoites under glucose-limiting conditions brought about a dramatic fall of ornithine decarboxylase activity, a key enzyme in polyamine biosynthesis. Levels of enzyme specific activity after 24 and 48 h of encystation represented only 11% and 1.3%, respectively, of those detected at the start of incubation. Induction of encystation in the presence of exogenously added polyamines resulted in a marked reduction in cyst formation. Thus, after 72 h of incubation, 1.0 mM putrescine, 1.0 mM spermidine or 0.5 mM spermine reduced encystation by 48 to 56%. Inhibition was enhanced to 70–73% in response to a two-fold increase in the concentration of either putrescine or spermine. Our results indicate that polyamine biosynthesis from ornithine is rapidly turned off at the onset of encystation.     

Effect of sodium butyrate on induction of ornithine decarboxylase activity in phytohemagglutinin-stimulated lymphocytes

Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin(PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [³H] thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other shortchain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.     

细菌鸟氨酸脱羧酶作用的研究进展

多胺是生物体内广泛存在的一类具有多种生物活性的低分子化合物,其合成的关键限速酶是鸟氨酸脱羧酶,鸟氨酸脱羧酶和多胺共同参与生物生长发育等重要生理过程。细菌鸟氨酸脱羧酶在结构上和真核生物略有不同,但是功能类似,其能通过促进多胺的产生发挥对细菌的调节作用。研究发现,细菌鸟氨酸脱羧酶也参与细菌对其他物种的作用,但对人体的作用尚不明确。因此,本文综述了国内外关于细菌鸟氨酸脱羧酶在促进细菌生长、适应环境、抗生素抗性和生物膜形成等方面的作用及相关机制,希望能对细菌鸟氨酸脱羧酶及其作用的后续研究提供一些信息与参考。     

Effect of experimental diabetes on ornithine decarboxylase activity of rat tissues

Measurements have been made of the activity of ornithine decarboxylase of liver, heart, kidney and brain in alloxan-diabetic and control rats. In all these tissues this enzyme had decreased markedly at four weeks after induction of diabetes. These results are discussed in relation to the hormonal control and cyclic nucleotide regulation of ornithine decarboxylase.     

Stable siRNA-mediated silencing of antizyme inhibitor: regulation of ornithine decarboxylase activity

Ornithine decarboxylase (ODC) is the rate-limiting enzyme involved in the biosynthesis of polyamines essential for cell growth and differentiation. Aberrant upregulation of ODC, however, is widely believed to be a contributing factor in tumorigenesis. Antizyme is a major regulator of ODC, inhibiting ODC activity through the formation of complexes and facilitating degradation of ODC by the 26S proteasome. Moreover, the antizyme inhibitor (AZI) serves as another factor in regulating ODC, by binding to antizyme and releasing ODC from ODC-antizyme complexes. In our previous report, we observed elevated AZI expression in tumor specimens. Therefore, to evaluate the role of AZI in regulating ODC activity in tumors, we successfully down-regulated AZI expression using RNA interference technology in A549 lung cancer cells expressing high levels of AZI. Two AZI siRNAs, which were capable to generate a hairpin dsRNA loop targeting AZI, could successively decrease the expression of AZI. Using biological assays, antizyme activity increased in AZI-siRNA-transfected cells, and ODC levels and activity were reduced as well. Moreover, silencing AZI expression decreased intracellular polyamine levels, reduced cell proliferation, and prolonged population doubling time. Our results directly demonstrate that downregulation of AZI regulates ODC activity, intracellular polyamine levels, and cell growth through regulating antizyme activity. This study also suggests that highly expressed AZI may be partly responsible for increased ODC activity and cellular transformation.     

In vivo induction of rat hepatic ornithine decarboxylase and plasminogen activator by 12-O-tetradecanoypphorbol 13-acetate

Rapid and substantial elevations in ornithine decarboxylase and plasminogen activator have been linked to tumor promotion in mouse epidermis and in vitro. Systemic administration of 12-O-tetradecanoylphorbol 13-acetate (TPA) rapidly increased both enzymic activities in rat liver. Pretreatment with either cycloheximide or actinomycin D attenuated both enzyme inductions. It is concluded that: (1) systemic TPA rapidly induces plasminogen activator and ornithine decarboxylase activities in rat liver; and (2) both inductions reflect de novo enzyme synthesis.     

Inhibition of epinephrine and gonadotropic hormone induced ornithine decarboxylase activity by phenoxybenzamine in the testis of immature rat

The effect of α and β adrenergic receptor blockers on epinephrine and gonadotropic hormone induced ornithine decarboxylase (ODC) activity in the testis of immature rats was studied. Intratesticular injection with phenoxybenzamine at 15 min before treatment with epinephrine or gonadotropic hormones blocked ODC activity. Similar injection with propranolol or practolol had no effect on ODC activity. These results show that α adrenergic receptors are involved in the action of epinephrine and gonadotropic hormones in the testis.     

Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells

Ornithine induced more than 36-fold the ornithine decarboxylase activity in confined Ehrlich ascites tumour cells after 3.5 h of continuous perifusion with 0.5 mM ornithine; arginine and glutamine also induced the activity 3- and 4-fold, respectively. The addition of cycloheximide or actinomycin D antibiotics to the perifusion medium confirmed that the regulation of the enzyme synthesis takes place at the level of translation. Perifusion in the presence of 0.5. mM ornithine and 55, 25, and 10 μM histamine suppressed the induction by 91, 53, and 35%, respectively. Similar results were obtained in the presence of serotonin. Histidine also showed inhibitory effect but 5 mM histidine was required to produce 21% inhibition; other basic amino acids were ineffective.     

Cyclic nucleotides in the denervated rat diaphragm and the effect of cyclic AMP on ornithine and adenosylmethionine decarboxylases

The concentration of cyclic AMP and cyclic GMP were measured in the denervated rat diaphragm at various times following unilateral phrenicectomy. Cyclic AMP concentration was raised by the second day after operation, reached a peak by the third day, followed by another increase at around 10 days. By contrast, cyclic GMP concentration was decreased within a day after denervation and remained below control levels at all subsequent times studied. Epinephrine in vitro produced a comparable increase in the concentration of cyclic AMP in both normal and denervated tissue. The concentration of adenosine appeared unchanged in the denervated diaphragm by comparison with its innervated contorl. Activity of ornithine decarboxylase was elevated in the diaphragms of rats treated with dibutyryl cyclic AMP, but this effect could also be achieved with sodium butyrate alone. Adenosylmethionine decarboxylase activity was unaffected after treatment with either compound. These observations and others discussed are taken to indicate a lack of direct relationship between cyclic AMP concentrations and the activity of the rate-limiting enzymes of polyamine biosynthesis in the rat diaphragm.     

The inhibition of the serum-stimulated increase of ornithine decarboxylase by ionophores and local anesthetics

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (l-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchine, vinblastine) or cations (5–10 mM K⁺, Na⁺ or Mg²⁺) abolishes this increase of ornithine decarboxylase activity (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212–219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six inonophores tested, valinomycin was the most potent one, with I₅₀ value (concentration that gives 50% inhibition of orthinine decarbocylase activity) of 6·10^?9 M. Dibucaine and tetracaine were also effective inhibitors at 10^?4?10^?5 M. The I₅₀ values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1·10^?6 M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.