Changes in ornithine decarboxylase and antizyme activities in developing mouse brain.

A macromolecular inhibitor to ornithine decarboxylase (ODC) present in mouse brain was identified as ODC antizyme [Fong, Heller & Canellakis (1976) Biochim. Biophys. Acta 428, 456-465; Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1858-1862] on the basis of kinetic properties, Mr and reversal of its inhibition by antizyme inhibitor. The brain antizyme, however, did not cross-react immunochemically with any of seven monoclonal antibodies to rat liver antizyme. ODC activity in mouse brain rapidly decreased after birth, in parallel with putrescine content, and almost disappeared by 3 weeks of age. Free antizyme activity appeared shortly after birth and increased gradually, whereas ODC-antizyme complex already existed at birth and then gradually decreased. Thus total amount of antizyme remained about the same throughout the developmental period in mouse brain. In addition to ODC-antizyme complex, inactive ODC protein was detected by radioimmunoassay in about the same level as the complex at 3 weeks of age. Upon cycloheximide treatment, both free ODC activity and ODC-antizyme complex rapidly disappeared, although free antizyme and the inactive ODC protein were both quite stable.     

Dietary induction of ornithine decarboxylase in male mouse kidney

In male mouse kidney, ornithine decarboxylase (ODC) is induced after feeding, and the induction depends on dietary protein content. 24 h after feeding with 50% casein-containing meal, ODC activity and amount of immunoreactive ODC protein increased more than 10-fold, ODC mRNA level increased 2-fold, and the ODC half-life extended 7-fold. The renal ODC induction after feeding is, therefore, due mainly to stabilization of ODC protein. Urinary excretion of putrescine increased in response to the ODC induction, but the renal polyamine contents scarcely changed. Consistently, the level of antizyme, a polyamine-inducible protein, determined as the ODC-antizyme complex level, scarcely changed after feeding, and the antizyme/ODC ratio in the kidney largely decreased, resulting in the stabilization of ODC protein. The present results suggest that the strong excretion system of the kidney for newly synthesized polyamines enables renal ODC escape from antizyme-mediated feedback regulation.     

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).     

Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.     

Ornithine decarboxylase antizyme in kidneys of male and female mice.

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.     

Two distinct mechanisms for ornithine decarboxylase regulation by polyamines in rat hepatoma cells.

Exogenous diamines and polyamines added to rat hepatoma (HTC) cells in culture rapidly decrease ornithine decarboxylase (ODC) activity. Previous evidence has suggested that these amines set either at the level of blocking new enzyme synthesis or by the induction of a non-competitive protein inhibitor, termed antizyme, which complexes with ODC to form an inactive complex. Wth the use of HMOA cells, a recently cloned rat hepatoma cell line that has a greatly stabilized ODC, it has been possible to demonstrate that 10(-5) M of exogenous putrescine blocks the increase in ODC activity, but unlike in the parent HTC cell line, without induction of the antizyme or formation of any inactive ODC-antizyme complex. However, complete blockade of ODC at 10(-2) M putrescine is effected by induction of antizyme and formation of the ODC-antizyme complex, as now evidenced by the isolation of the active enzyme and antizyme components after Sephadex column chromatography in the presence of 250 mM NaCl. These findings indicate clearly that two polyamine-regulatory mechanisms for ODC exist and are separable in this cell line.     

Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver.

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.     

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.     

Destabilization of ornithine decarboxylase by transfected antizyme gene expression in hepatoma tissue culture cells.

The degradation of ornithine decarboxylase (ODC) is stimulated by polyamines in a protein synthesis-dependent manner. It has been suggested that antizyme, an ODC-inhibiting protein induced by polyamines, is involved in the process of polyamine-stimulated ODC decay. In this study, we investigated the direct effect of antizyme on ODC decay in hepatoma tissue culture (HTC) cells. A truncated rat antizyme cDNA, Z1, was inserted into an expression vector at a site under the control of a glucocorticoid-inducible promoter and transfected into HTC cells. In the transfected cells dexamethasone increased the amount of Z1 mRNA and induced active antizyme in the absence of exogenous polyamines. When dexamethasone was added to cells with a high level of ODC, rapid decays of ODC activity and protein were elicited after a lag time. Cycloheximide abolished the effect of dexamethasone. These effects of dexamethasone were not observed in control HTC cells transfected with the chloramphenicol acetyltransferase gene. This study indicated that, once induced, antizyme stimulated ODC degradation independently of polyamines and strongly supported our previous hypothesis that the ODC decay-accelerating action of polyamines is mediated by antizyme.     

A macromolecular inhibitor of the antizyme to ornithine decarboxylase.

A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, 'antizyme inhibitor', was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.     

Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay

Antibodies were produced in rabbits to homogeneous mouse kidney ornithine decarboxylase and used to determine the amount of this protein present in kidney extracts by a competitive radioimmunoassay procedure. The labeled ligand for this assay was prepared by reacting renal ornithine decarboxylase with [5-3H] alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The sensitivity of the assay was such that 1 ng of protein could be quantitated and the binding to ornithine decarboxylase of a macromolecular inhibitor (antizyme) or alpha-difluoromethylornithine did not affect the reaction. It was found that treatment of female mice with testosterone produced a 400-fold increase in ornithine decarboxylase protein in the kidney within 4-5 days. Exposure to cycloheximide or to 1,3-diaminopropane led to a rapid disappearance of the protein which paralleled the loss of enzyme activity. There was no sign of any immunoreactive but enzymatically inactive form of mouse kidney ornithine decarboxylase under any of the conditions investigated. The results indicate that fluctuations of the enzyme activity in this organ are mediated via changes in the amount of enzyme protein rather than by post-translational modifications or interaction with inhibitors or activators.     

Antizyme to ornithine decarboxylase is present in the liver of starved rats.

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.     

Existence of antizyme and ornithine decarboxylase-antizyme complex in RK13 kidney cells

It has been reported that 'antizyme', a protein inhibitor of ornithine decarboxylase (ODC) induced by its product, is not found in rat or mouse kidney. We determined whether antizyme was present in rabbit kidney cells (RK13) in culture. Antizyme could be induced in these cells by putrescine treatment, a substantial portion being in the particulate fraction in contrast with hepatic antizyme. Furthermore, ODC-antizyme complex was present even in untreated cells. Pretreatment of cells with putrescine increased the relative amount of ODC-antizyme complex and accelerated decay of ODC. These results support the ubiquitous existence of antizyme and its role in ODC degradation.     

Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein.

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992). We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.