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PubMed1994

A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme.

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamine-induced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC-Az binding site of ODC and the ODC binding site of ODC-Az, acceleration in ODC degradation also requires the previously characterized carboxyl-terminal destabilizing segment of ODC and a specific segment of ODC-Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC-Az binding site is stable under basal degradation conditions. This, together with the ability of anti-(ODC-Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC-Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.

Amino Acid Sequence↗
PubMed2000

The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.

Adenosylmethionine Decarboxylase↗
PubMed1996

Contrasting effects of thioacetamide-induced liver damage on the brain uptake indices of ornithine, arginine and lysine: modulation by treatment with ornithine aspartate.

The dibasic amino acids arginine (ARG), ornithine (ORN) and lysine (LYS) are transported by a common saturable transporter (system gamma +) at the blood-brain barrier (BBB). In the present study we compared the brain uptake index (BUI) for radiolabelled ORN, ARG and LYS in control rats and in rats treated with thioacetamide (TAA) to induce hepatic encephalopathy (HE). Some animals received i.v. ornithine aspartate (OA), a drug structurally related to the gamma + substrates that ameliorates neurological symptoms following liver damage by improving detoxification of ammonia in peripheral tissues: the compound was administered either by continuous infusion for 6h at a concentration of 2 g/kg (final blood concentration ranging from 0.19-0.5 mM), or as a 15 sec. bolus together with the radiolabelled amino acids, at a concentration of 0.35 mM. TAA treatment resulted in a delayed and progressive increase of BUI for ORN, to 186% of control at 7d post-treatment and to 345% of control at 21d post-treatment, when despite sustained liver damage, HE symptoms were already absent. In contrast, the BUI for ARG decreased to 30% of control at 7d post-treatment and remained low (42% of control) at 21d post-treatment. A 6h infusion of OA to untreated rats resulted in a reduction of the BUI for ARG and ORN to 51% and 62% of the control levels, respectively. Reductions of a similar magnitude were noted with both amino acids following the 15 sec OA bolus, indicating direct interaction of OA with the transport site in both cases. OA administered by either route abolished the enhancement of BUI for ORN, but did not further inhibit the BUI for ARG in the TAA-treated animals. The results indicate that some as yet unspecified factors released from damaged liver either modify the structure or conformation of the gamma + transporter at the BBB from the normally ARG-preferring to the ORN-preferring state, or activate (induce) a different transporter specific for ORN which is normally latent.

Animals↗
PubMed2002

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.

Animals↗
PubMed1985

Release of hepatic mitochondrial ornithine transcarbamylase into the circulation in D-galactosamine-treated rats. Identification of serum ornithine transcarbamylase as the intact form of the mitochondrial enzyme.

A single injection of D-galactosamine into rats caused acute liver cell injury, and the activity of ornithine transcarbamylase in the serum increased about 600-fold as compared with that in the normal serum. Some properties of the serum enzyme from galactosamine-treated rats have been studied together with those of the mitochondrial enzyme in liver. Both the enzyme activities gave similar pH profiles, showing an optimum of pH 8.5. Apparent Km values of the serum enzyme for ornithine under the standard conditions at pH 7.4 and pH 7.7 were 1.59 mM and 0.94 mM, respectively, and those of the mitochondrial enzyme were 1.69 mM and 0.97 mM, respectively. The Km value of the serum enzyme for carbamyl phosphate was 0.34 mM, which is similar to that of the mitochondrial enzyme. The mitochondrial enzyme was purified 78-fold to homogeneity with a 45% yield by ammonium sulfate fractionation, heat treatment, 2nd ammonium sulfate fractionation, and phenyl-Sepharose CL-4B and CM-Sephadex C-50 column chromatographies. The specific activity of the purified enzyme was 282 mumol of citrulline formed per mg of protein per min at 37 degrees C. The mitochondrial and serum enzymes have a molecular weight of 115,000 as determined by Sephacryl S-300 gel filtration. Antibody specific for the mitochondrial enzyme was raised, and the immunological properties of the serum enzyme were examined. In immunoinhibition experiments, a decrease of the serum enzyme activity as well as the mitochondrial enzyme was observed on increasing the amount of the antibody.

Animals↗
PubMed1982

Ornithine decarboxylase and polyamines in tissues of the neonatal rat: effects of alpha-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase.

To evaluate the role of ornithine decarboxylase (ODC) and the polyamines in tissue growth and development, neonatal rats were given daily injections of alpha-difluoromethylornithine, a specific, irreversible inhibitor of ODC. Enzyme activity in brain, heart and kidney was effectively inhibited, leading to prompt reductions in putrescine levels which were apparent throughout the 4-week period of drug treatment. Deficits in spermidine levels appeared within several days and remained significant in all three tissues, although some recovery toward control levels was apparent after 2 weeks postnatally. Spermine levels were not decreased and in some cases were actually increased during the course of alpha-difluoromethylornithine administration; assessment of total organ content of spermine or total polyamines per organ (putrescine + spermidine + spermine) indicated that the tissues were still actively increasing their net polyamine content despite continued inhibition of ODC. Growth of the kidney and brain were affected within several days of commencing alpha-difluoromethylornithine treatment, well before the onset of body weight or heart weight deficits. By 14 days of age and thereafter, animals displayed delayed eye-opening, deficient fur growth and shorter body length. These data suggest that the ODC/polyamine system does serve as a modulator of tissue growth during neonatal mammalian development and that differences exist among various tissues in the degree and time course of dependence of growth on polyamines, particularly putrescine and/or spermidine.

Animals↗
PubMed1992

Ornithine decarboxylase and ornithine decarboxylase-inhibiting activity in rat thymocytes.

Isolation of thymocytes from rat thymus resulted in the disappearance of the high activity of ornithine decarboxylase (ODC) that characterizes the thymus of young rats, together with the appearance of an antizyme-like ODC inhibiting activity, which showed a chromatographic profile that resembled that of dexamethasone-treated rat thymus. Omission of serum or addition of dexamethasone or spermidine did not affect appreciably the extent of the antizyme-like activity. On the other hand, a variety of hormonal effectors, i.e. insulin, glucagon, adrenalin and T3, as well as the phorbol ester, PMA or the mitogen, concanavalin A (Con A) induced ODC activity in cultured thymocytes together with the disappearance of the antizyme-like activity. A paradoxical, transient induction of ODC was caused by the transcriptional inhibitor, actinomycin D. Complexed ODC was detected in rat thymus, but not in thymocytes, either quiescent or stimulated by mitogens. These results indicate that thymic lymphocytes can express either ODC activity or its inhibitor depending on the hormonal and proliferative status of the cells.

Animals↗
PubMed1991

A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency.

We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the OTC gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.

Alleles↗
PubMed2002

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.

Animals↗
PubMed1985

Ornithine transcarbamylase and disaccharidase activities in damaged intestinal mucosa of children--diagnosis of hereditary ornithine transcarbamylase deficiency in mucosa.

Ornithine transcarbamylase (OTC) and disaccharidase activities were measured in the intestinal mucosa from 182 children. Sixty-nine had normal mucosa, whereas the others had different degrees of mucosal damage. Brush border disaccharidases are significantly decreased in all degrees of villous atrophy. In contrast, OTC is not affected in moderate atrophy and only slightly decreased in severe atrophy. Consequently, the OTC-to-lactase ratio increases with the degree of atrophy and permits discrimination between normal and damaged mucosa. The assay of OTC activity in intestinal mucosa for the diagnosis of hereditary deficiency in male hemizygote patients generally provides nonambiguously low results, whereas in heterozygote females the amount of residual activity is in the range of the results found in damaged mucosa.

Child↗
PubMed1989

Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene.

To define the molecular basis for the TaqI site alteration in the ornithine transcarbamylase (OTC) (E.C.2.1.3.3) gene of a female patient with mild OTC deficiency, we used a combination of genomic amplification followed by direct sequencing and oligodeoxyribonucleotide hybridization. We obtained evidence for a C-to-T substitution in exon 5 (codon 141) of this gene. This mutation generates a stop codon, in place of Arg, at amino acid 109 of the mature OTC protein. The mutation arose, de novo, in a germ cell of one of the parents.

Adolescent↗
PubMed1996

Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase.

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and nucleotide sequence analysis of mRNA for human kidney ornithine aminotransferase. An examination of ornithine aminotransferase isozymes between liver and kidney.

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3'- and 5'-untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N-terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln-35 and Gly-36.

Amino Acid Sequence↗
PubMed1989

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.

Animals↗
PubMed1998

Neuroprotective role of ornithine decarboxylase activation in transient focal cerebral ischaemia: a study using ornithine decarboxylase-overexpressing transgenic rats.

Nuclear magnetic resonance imaging (MRI) was used to study dynamics of maturation and the size of ischaemic stroke lesions in rats with greatly increased activity of ornithine decarboxylase (ODC). Syngenic rats, either with or without chronic pre-ischaemic treatment with an ODC inhibitor, alpha-difluoromethylornithine (DFMO), as well as ODC-overexpressing transgenic rats were subjected either to transient middle cerebral artery (MCA) occlusion or permanent occlusion of the cortical branch of MCA. The two models were chosen to assess the role of ODC activity in damage caused by ischaemia and reperfusion, respectively. Diffusion of water was quantified by means of the trace of the diffusion tensor (D(av) = 1/3 Trace D) to assess the extent of energy failure and cytotoxic oedema, whereas the spin-spin relaxation time (T2) was used as a quantitative indicator of irreversible damage by MRI. Exposure to transient MCA occlusion resulted in significantly smaller stroke lesions in the ODC-overexpressing transgenic (246+/-14 mm3) than in syngenic (320+/-9 mm3) or DFMO-treated (442+/-63 mm3) rats as determined 48 h after the occlusion. The differences in sizes were due to smaller lesions in the cortical tissue (transgenic vs. syngenic) or both in cortical and striatal regions (transgenic vs. DFMO-treated animals). The degree of irreversible oedema was greater in DFMO-treated rats than in syngenic or transgenic animals indicating accelerated development of a permanent damage in the absence of ODC induction. Cortical infarct following permanent MCA occlusion developed faster in the DFMO-treated than in syngenic or transgenic rats as the lesion sizes at 10 h were 26.2+/-4.3 mm3, 14.2+/-2.3 mm3 and 12.3+/-1.9 mm3, respectively. However, the stroke volumes by 48 h were not statistically different in the three animal groups. The present data demonstrate that ODC activation is an endogenous neuroprotective measure in transient cerebral ischaemia.

Animals↗
PubMed2006

Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

Amino Acid Sequence↗
PubMed1990

Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase.

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

Alcaligenes↗
PubMed1984

Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli. Molecular cloning of rat ornithine transcarbamylase.

A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA [Horwich, A. L., Kraus, J.P., Williams, K., Kalousek, F., Königsberg, W. & Rosenberg, L.E. (1983) Proc. Natl Acad. Sci. USA, 80, 4258-4262]. Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence [Lusty, C., Jilka, R. L. & Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036]. A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted.

Amino Acid Sequence↗