Absence of inactivation or phosphorylation of ornithine decarboxylase by nuclear protein kinase NII and of immunological cross-reactivity between RNA polymerase I and ornithine decarboxylase

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.     

Histochemical localization of ornithine decarboxylase with a labelled suicidal enzyme inhibitor

We describe a new technique for cytochemical localization of ornithine decarboxylase by the use of a synthesized conjugate of rhodamine bound to α-difluoromethylornithine a suicidal inhibitor of the enzyme. The labelled inhibitor retained its specificity and irreversibility towards ornithine decarboxylase inhibition. Using this technique we have localized the enzyme in specific regions of the developing rat cerebellum. This novel technique may be generally applicable to other enzymes.     

Inhibition of ornithine decarboxylase induces embryonal carcinoma cell differentiation

Murine embryonal carcinoma cells can be induced to differentiate in vitro by various physical and chemical means. We report here that inhibition of ornithine decarboxylase activity with a specific enzyme-activated inhibitor, alpha-difluoromethylornithine, can induce differentiation in embryonal carcinoma cells. The differentiated phenotype can be distinguished from undifferentiated embryonal carcinoma cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the levels of cellular polyamines may play a role in embryonal carcinoma cell differentiation.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

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.     

Plasmodium berghei: inhibitors of ornithine decarboxylase block exoerythrocytic schizogony

DL-alpha-difluoromethylornithine and DL-alpha-monofluoromethyldehydroornithine methyl ester, inhibitors of ornithine decarboxylase, blocked exoerythrocytic schizogony of Plasmodium berghei in mice and in cultured human hepatoma cells. These effects were reversed by exogenous administration of the polyamine, spermidine. The antimalarial drug, primaquine, the side chain of which is structurally analogous to a natural polyamine, did not enhance the activity of alpha-difluoromethylornithine or alpha-monofluoromethyldehydroornithine methyl ester. These results extend previous observations that polyamines influence the malaria parasite's schizogony outside the red blood cell but not within it.     

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.     

Isoproterenol-induced responses in the regional polyamine metabolism of the rat heart

Rats were treated with a single dose of isoproterenol (25 mg/kg s.c.) and the levels of polyamines determined in various parts (right ventricle, basis, medial part and apex of the left ventricle) of the heart 24, 48 and 72 hours after the injection. The isoproterenol treatment produced marked alterations in the concentrations of cardiac polyamines. The most apparent changes were seen in the apex and medial part of the left ventricle where spermidine concentration exhibited a biphasic response with peaks at 24 and 72 hours. In the basis of the heart the spermidine concentration was significantly elevated only at 24 hours. In the right ventricle the spermidine level was significantly higher than control at 72 hours. Spermidine/spermine ratio was augmented in all cardiac tissues examined over the 72-hour period. Results appear to show that the isoproterenol-induced alterations in cardiac polyamine metabolism were not uniformly distributed in the various regions of the heart.     

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.     

Reversal of antizyme-induced inhibition of ornithine decarboxylase by cations in barley seedlings

Ornithine decarboxylase (ODC) forms a stable complex with its antizyme (Az), a non-competitive protein inhibitor of ODC. The complex formation of ODC with Az occurs very rapidly and is dissociated by high salt concentrations e.g., 10% ammonium sulfate. When ODC and Az were mixed in the presence of increasing concentrations of Mg²⁺, a relief of ODC inhibition by Az was obtained. Complete relief of inhibition occurred at 2.0 mM of MgCl₂. Other bivalent cations Ca²⁺, Ba²⁺, Co²⁺, Mn²⁺, Zn²⁺ as well as the monocations Na⁺ and K⁺ caused similar effect. The polyamines putrescine, spermidine and spermine also caused relief of the in vitro inhibition of ODC by Az. Therefore, the in vivo inactivation of ODC by forming the ODC-Az complex is dependent on the intracellular amounts of salt and polyamines.     

Phosphorylation of rat heart ornithine decarboxylase by type-2 casein kinase

Highly purified preparations of rat heart ornithine decarboxylase are readily phosphorylated by rat liver type-2 casein kinase-TS at the same 54 KDa protein band which is also radiolabeled by 3H-DFMO. The reaction, which is stimulated by polylysine leads to the incorporation of up to 0.8 mol P/mol ornithine decarboxylase at seryl residue(s) included in a single 8.6 KDa CNBr fragment. Partially purified preparations of ornithine decarboxylase contain a type-2 casein kinase which promotes the phosphorylation of ornithine decarboxylase at the same CNBr fragment affected by rat liver casein kinase-TS.     

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Background

Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.

Methods

Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.

Results

The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.

Conclusions

These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.

General significance

This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.     

Generating knock-in parasites: integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani

Leishmania null mutants created by targeted gene replacement are typically complemented with chimeric episomes harboring the replaced gene in order to validate that the observed phenotype is due to the specific gene deletion. However, the current inventory of available episomes for complementation of genetic lesions in Leishmania is unstable in the absence of drug selection, and levels of gene expression cannot be controlled, especially in vivo. To circumvent this impediment, a strategy to re-introduce the targeted gene into the original chromosomal locus to generate “knock-in” parasites within selectable null backgrounds has been developed. A genomic fragment encompassing the ornithine decarboxylase locus and lacking heterologous DNA sequences was transfected into ornithine decarboxylase-deficient Leishmania donovani. The construct randomly integrated into either chromosomal allele by homologous recombination restoring polyamine prototrophy and revealing that LdODC was functionally expressed in the knock-in clones. This strategy offers a mechanism for complementing a genetic lesion amenable to positive selection in a manner that facilitates stable gene expression from its original locus in the absence of continuous drug pressure.     

Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p>     

Ornithine decarboxylase protein diversity and activity modulation in HTC cells

Ornithine decarboxylase of HTC cells was chromatographically separated into three ionically distinct but kinetically similar forms of this protein. The sequential appearance of these ornithine decarboxylase species during enzyme induction, and the accumulation of normally minor species under conditions that stabilize this enzyme, suggest that these represent modifications that are associated with the extremely rapid turnover of this protein $\text{in vivo}$. These forms may also be differentially active or unequally distributed $\text{in vivo}$ as indicated by the selective inactivation of one of the forms by short exposure to α-difluoromethylornithine.     

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0 mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 °C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC₅₀ values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0 mg/mL, respectively.     

The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODC-antizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2, and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC, GM853 was a stable protein that was not able to decarboxylate l-ornithine or l-lysine and that did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on l-leucine (Km = 7.03 × 10^? 3 M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, l-valine and l-isoleucine were poor substrates of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian species, including primates, underlines the possible biological significance of this new enzyme. In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as leucine decarboxylase (Ldc, LDC).     

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for -receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic -selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific -antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, -opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.     

Polyamine biosynthesis in Phytomonas: Biochemical characterisation of a very unstable ornithine decarboxylase

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5′-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.