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.

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.

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.

The synthesis of S-adenosylmethionine (AdoMet) decarboxylase was studied by translating the rat prostate mRNA for this enzyme in a reticulocyte lysate. The protein was formed as a precursor of Mr 37,000, which was converted into the enzyme subunit of Mr 32,000 in the lysates. The presence of putrescine had no effect on the synthesis of the precursor of AdoMet decarboxylase, but accelerated its conversion into the enzyme subunit. Spermidine, spermine, decarboxylated AdoMet, AdoMet and methylglyoxal bis(guanylhydrazone) were not able to substitute for putrescine in this effect. These results indicate that, in addition to its direct activation of mammalian AdoMet decarboxylase, putrescine could increase the amount of the enzyme by increasing its production.

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Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.Increased rates of synthesis of the polyamines, putrescine, spermidine and spermine, accompany a variety of cellular stimuli, including responses to growth factors, tumor promoters and hormones [I, 21. These increases are required for cell proliferation [2-41. The first step of polyamine biosynthesis is catalyzed by ornithine decarboxylase (ODC), which is also a major site of regulation of the pathway. The cellular levels of ODC activity and protein are elevated rapidly in response to stimuli. The rapid kinetic course of ODC induction is due, at least in part, to the rapid turnover of this protein. Recently, cloning of cDNAs coding for ODC has facilitated analysis of the behavior of ODC mRNA [5-81. Elevated levels of ODC mRNA accompany induction of the enzyme in at least three systems: serum-stimulated mouse fibroblasts [7], kidneys from androgen-treated mice [6 -81 and rat pheochromocytoma cells treated with nerve growth factor [9]. In this paper we show that induction of ODC during lymphocyte mitogenesis results from both increases in the level of ODC mRNA and in the efficiency of its translation. This latter mode of control is due to an increase in the fraction of ODC mRNA associated with polysomes, which occurs after the elevation of ODC mRNA level. MATERIALS AND METHODS Cell cultureBovine lymphocytes were purified from suprapharyngeal lymph nodes as previously described [lo]. Cells were stimulat- RNA purification, electrophoresis and hybridizationTotal RNA was purified from cells at various times after ConA addition by the guanidium isothiocyanate method of Chirgwin et al. [ll]. RNA from sucrose gradients was prepared by incubating each gradient fraction with SDS and proteinase K (final conce...

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