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Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. We have shown that its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Here we report the identification of the protease responsible for ODC degradation. Using a cell-free degradation system, we demonstrate that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis.

We have isolated from mouse FM3A cells a variant cell line, termed EXOD-1, that overproduces ornithine decarboxylase (ODC). The cells were resistant to alpha-difluoromethylornithine, an irreversible inhibitor of the enzyme, and produced the enzyme protein to the extent of approx. 3-6% of total cytosolic protein. The rate of ODC synthesis in this cell line accounted for 25-50% of the rate of total protein synthesis. The amounts of the ODC gene and its mRNA in the variant cells were both about 60 times as much as those in wild-type FM3A cells. Upon removal of the inhibitor, the growth of the ODC-overproducing cells was stimulated approx. 2-fold. Under these conditions, the rate of ODC synthesis increased about 4-fold on day 1 and then decreased to near the original level by day 3. The amount of ODC mRNA increased about 1.7-fold on day 1 and 2.5-fold on day 3. No correlation was observed between changes in ODC synthesis rate and in ODC mRNA content, suggesting a translational repression of ODC mRNA due to accumulation of polyamines. In fact, the cellular contents of putrescine and spermidine markedly increased and that of spermine inversely decreased during the same period. Pulse-chase experiments showed that the accumulation of putrescine and spermidine also elicited a rapid degradation of ODC. Excess amounts of newly synthesized putrescine and cadaverine were excreted into the medium, whereas spermidine, spermine and acetylated polyamines were undetectable there. We conclude that ODC regulation upon removal of the inhibitor is dependent on at least three steps, namely the level of mRNA, the translational efficiency of mRNA and the stability of the enzyme, the last two of which are involved in cellular polyamines.

We investigated the mechanisms underlying the marked induction of ornithine decarboxylase (ODC) activity by hypotonic treatment and its rapid decay upon reversal to isotonicity in primary cultures of adult rat hepatocytes. Upon hypotonic treatment, ODC synthesis rate increased progressively whereas the amount of ODC mRNA increased only about twofold. In addition, ODC was stabilized severalfold. ODC activity rapidly decreased upon restoration of isotonicity, owing to immediate and nearly complete suppression of ODC synthesis and 3-6-fold stimulation of ODC decay. The stimulation of ODC decay caused by restoration of isotonicity was mostly independent of time and protein synthesis. ODC decay was also stimulated by putrescine, even under hypotonic conditions, depending on time and new protein synthesis. Restoration of isotonicity and putrescine treatment together caused a synergistic stimulation of ODC decay, confirming that these act by different mechanisms.

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 3.5·10 5 -fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10 6 nmol CO 2 /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.

A monoclonal antibody was obtained against rat liver ornithine decarboxylase by using hybridoma technology with a small amount of partially purified enzyme. The antibody, IgG1 of kappa-type, was affinity-purified to homogeneity from culture supernatants of hybridoma cells. While the antibody had no inhibitory effect on ornithine decarboxylase activity when tested alone, it precipitated up to 87 units (60 ng) of the enzyme per microgram in the presence of formalin-fixed Staphylococcus aureus Cowan I bacteria. Immunoadsorption on a column of the monoclonal antibody-Sepharose 4B was shown to be useful for the removal of ornithine decarboxylase from antizyme inhibitor preparations, an essential procedure for the accurate assay of either ornithine decarboxylase-antizyme complex or antizyme inhibitor. It was also shown that antizyme could be affinity-purified by using a column of the monoclonal antibody-Affi-Gel 10 to which ornithine decarboxylase had been bound.

Ornithine decarboxylase (ODC) mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.

To pursue the mechanism underlying dietary induction of ornithine decarboxylase (ODC) activity in rat liver, changes in free-polysome-associated messenger RNA (mRNA) activity for the enzyme were studied in rats fed diets containing either casein or zein. The enzyme activity increased 50- to 100-fold in 4 h after feeding a 50% casein diet. Changes in the amount of immunoreactive ODC protein roughly paralleled changes in the enzyme activity. Dietary casein was found to cause a several fold increase in polysomal mRNA activity coding for this enzyme, which preceded the increase in catalytic activity. Therefore, the induction of hepatic ODC by casein feeding is due partly to an increase in the activity of its mRNA associated with free polysomes and partly to some translational and/or posttranslational control. On the other hand, feeding the zein diet hardly increased ODC activity, immunoreactive ODC protein or free-polysomal ODC-mRNA activity. This indicates that adequate supply and balance of amino acids are required both for the increase in polysome-associated ODC-mRNA activity and for the translational and/or posttranslational mechanism(s) during the induction of hepatic ODC activity.

In rats fed ad libitum, a marked circadian rhythm with a peak at night was observed in the hepatic level of ornithine decarboxylase (ODC) [EC 4.1.1.17], the enzyme for the first step of polyamine synthesis. A similar rhythm was found in the hepatic content of putrescine, but not of spermidine or spermine. The mitotic activity of the liver also exhibited a clear rhythm with a peak in the daytime. The rhythms of both ODC and mitosis were generated by cyclic ingestion of proteinous food, since the peaks shifted when rats were meal-fed and both activities disappeared on starvation or protein deprivation. The close parallel between the rhythms suggested that synthesis of polyamine, especially that of putrescine, was a prerequisite for the rhythmic growth of liver. The dietary induction of hepatic ODC depended on the nutritive value of dietary protein; zein or gelatin was effective only when supplemented with limiting amino acids and there was a good correlation between the hepatic ODC level and the relative growth rate.

When rat liver homogenate or its postmitochondrial supernatant was incubated with L-cysteine, but not D-cysteine, ornithine decarboxylase (ODC) lost more than half of its catalytic activity within 30 min and, at a slower rate, its immunoreactivity. The inactivation correlated with production of H2S during the incubation. These changes did not occur in liver homogenates from vitamin B6-deficient rats. A heat-stable inactivating factor was found in both dialysed cytosol and washed microsomes obtained from the postmitochondrial supernatant incubated with cysteine. The microsomal inactivating factor was solubilized into Tris/HCl buffer, pH 7.4, containing dithiothreitol. Its absorption spectrum in the visible region resembled that of Fe2+ X dithiothreitol in Tris/HCl buffer. On the other hand FeSO4 inactivated partially purified ODC in a similar manner to the present inactivating factor. During the incubation of postmitochondrial supernatant with cysteine, there was a marked increase in the contents of Fe2+ loosely bound to cytosolic and microsomal macromolecules. Furthermore, the content of such reactive iron in the inactivating factor preparations was enough to account for their inactivating activity. These data suggested that H2S produced from cysteine by some vitamin B6-dependent enzyme(s) converted cytosolic and microsomal iron into a reactive loosely bound form that inactivated ODC.

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.

Tissue levels of ornithine decarboxylase (ODC) are under negative feedback regulation by polyamines. Thus, exogenously added polyamines cause a rapid decrease of ODC activity. Recent studies in various laboratories have indicated that the rapid decay of ODC activity caused by polyamines is due both to suppression of ODC synthesis (1–3) and to acceleration of ODC degradation (1, 2, 4) and that a new protein synthesis is needed for the latter effect (2). Antizyme was discovered in 1976 by Canellakis and his collaborators as a unique protein inhibitor of ODC induced by polyamines (5, 6). Since then, antizyme has been shown to be present in various tissues and cells as a free form and/or a complex with ODC (7). Previous studies in this and other laboratories have led to a hypothesis that antizyme not only inhibits ODC activity but also accelerates its degradation in a recycling manner (8–10). The strongest evidence for that hypothesis was the existence of a good correlation between ODC decay rate and antizyme/ODC ratio in HTC cells (9). In the present paper, we report the results of our recent studies that support the above hypothesis. We also present some of our results on the cloning of cDNAs for rat liver antizyme.

Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 microM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration of ODC degradation.

A single injection of thioacetamide into starved rats induced a 40- to 100-fold increase in hepatic ODC activity. However, immunotitratable ODC protein increased by only 5-fold because of the presence of significant amounts of inactive ODC protein in the liver of untreated starved rats. Polysomal ODC-mRNA activity also increased only 5-fold, a significant amount being present in control liver. Furthermore, the peak of polysomal ODC-mRNA activity preceded that of ODC activity or ODC protein by several hours. These results indicate that the enzyme induction is due not only to increase in polysomal ODC-mRNA activity, but also to some translational and/or post-translational regulation. Exogenously administered diamines or polyamines cause rapid decay of ODC activity and induce antizyme that binds to ODC and inactivates it. Another protein factor, antizyme inhibitor, was found in the liver of thioacetamide-treated or protein-fed rats. Antizyme inhibitor binds to antizyme and reactivates ODC in the ODC-antizyme complex. A small, but significant, amount of antizyme was found in the liver of starved rats. Only small amounts of ODC-antizyme complex were detected in rat liver and cultured hepatocytes, even during the period of rapid ODC decay caused by exogenously added diamines. On the other hand, the complex was present in HTC cells and more especially in ODC-stabilized HMOA cells, even under physiological conditions. On addition of 10(-2) M putrescine, the amount of complex first increased and then decreased in both types of cells. Decay of total ODC activity (free plus complexed ODC) was more rapid with putrescine than with cycloheximide. These results suggest that antizyme plays an essential role in the degradation of ODC by a catalytic effect both in the presence and absence of exogenous putrescine and that antizyme inhibitor stabilizes ODC by removing antizyme.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.