By Positive and Negative Effectors (Antizyme/Post-Translational Control/Ornithine Decarboxylase Inhibitor/Ornithine Decarboxylase Activator) DIMITRI A

Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 4699-4703, October 1978 Biochemistry

Modulation of ornithine decarboxylase activity in Escherichia coli by positive and negative effectors (antizyme/post-translational control/ornithine decarboxylase inhibitor/ornithine decarboxylase activator) DIMITRI A. KYRIAKIDIS, JOHN S. HELLER, AND E. S. CANELLAKIS Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Philip Siekevitz, July 3, 1978

ABSTRACT Two effectors of ornithine decarboxylase cellular levels of these nucleotides; this suggested a corre- (ODC; L-ornithine carboxy-lyase, IC 4.1.1.17) have been extracted from an ODC- (speC) mutant, Escherichia coli MA sponding lack of regulation of ODC activity by these nucleo- 255. One of these is an ODC inhibitor (M, 15,000 ± 2000) that tides in vivo in this strain of E. coli. is labile to t sin; its activity increases 20-fold in response to Of the various bacterial mutants available with mutations increased polyamine levels in the growth medium. It has addi- in genes related to ODC (9), we have used the bacterial mutant tional characteristics similar to those of the ODC antizyme of strains developed by Cunningham-Rundles and Maas (10) and eukaryote cells: it is a noncompetitive inhibitor of ODC;- the have identified a nondialyzable inhibitor as well as a macro- complex formed between ODC and the ODC inhibitor can be dissociated with salt to provide active ODC and active ODC molecular activator of ODC in E. coli. Their intracellular levels inhibitor; furthermore, this E. coli ODC inhibitor is inhibitory change during the growth of E. coli in concert with ODC ac- to eukaryote ODC. A thermostable nondialyzable factor that tivity and in response to the putrescine concentration in the activates ODC in vitro has also been extracted from MA255; medium. increased polyamine levels in the growth medium caused a 1.6-fold increase in the activity of this ODC activator. Effectors with comparable activities have also been identified in the MATERIALS AND METHODS parent ODC+ (speC+) strain MA197. The fluctuations of the Materials. Putrescine and spermidine were obtained from intracellular levels of these two ODC effectors during the Aldrich (Milwaukee, WI) and purified according to Heller et growth of E. coli MA255 have been related to the temporal changes of the activity of ODC in the parent ODC+ MA197 al. (11). DL-[1-14C]Ornithine (32.2 Ci/mol) was obtained from strain. The mode of interaction of these three macromolecules, New England Nuclear (Boston, MA). Trypsin, trypsin inhibitor, as reflected in the changes of the activity of ODC, appears to pyridoxal phosphate, Tris, and dithiothreitol were from Sigma be complex. The results suggest that ODC activity may be con- (St. Louis, MO). Bactotryptone and yeast extract were from trolled post-translationally by macromolecules that act as pos- Difco Laboratories (Detroit, MI). itive and negative effectors and whose levels fluctuate in re- Bacterial Strains and Media. E. coli, wild type K-12, strain sponse to the concentration of the end products of the reaction. 5073, MA135 (his-, trp-, thi-, proA, speB, argE), MA163 (thr-, leu-, thi-, his-, speA), MA197 (serA, thr-, leu-, thi-, We reported (1, 2) that the activity of ornithine decarboxylase speB), MA255 (thr-, leu-, thi-, speB, speC), and AB1203 (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) can be inhibitied (thi-, iv-, argE) were obtained from the Genetic Stock Center by a specific protein, the ODC antizyme, and proposed (3) that of Yale University. The E. coli (except MA255) were grown, macromolecular activators of ODC may also exist to balance with aeration at 370, in minimal medium E as described by the inhibitory action of the ODC antizyme. The appearance Vogel and Bonner (12) supplemented with 1 ml of trace ele- of the ODC antizyme, a noncompetitive inhibitor of ODC in ments per liter (13), 0.2% D-glucose, 50 mg of amino acids per eukaryote cells, occurred in response to high concentrations of liter, and 20 ,g of thiamine per liter. The MA255 cells were putrescine, spermidine, and spermine (1, 2). The term "anti- grown in Luria's medium (10 g of Bactotryptone, 5 g of yeast zyme" was suggested for this type of macromolecular inhibitor extract, and 0.5 g of NaCl per liter) at pH 7.0, normally in the of an enzyme, whose synthesis is induced by the product of the presence of 1.13 mM putrescine and 0.68 mM spermidine. reaction it inhibits (2). We now provide evidence for the exis- Usually 1 ml of E. coli (approx. 108 cells per ml) stored at -200 tence of two macromolecules in extracts of Escherichia coli, one in 5% (vol/vol) glycerol was inoculated into 500 ml of growth that activates and one that inhibits ODC. medium; after 12 hr of growth, this was added to 8 liters of Two different ODCs exist in E. coli, a biodegradative en- growth medium and the growth was continued. Unless other- zyme induced by growth at low pH and a biosynthetic enzyme wise noted, cells were harvested in midlogarithmic phase, detectable by growth at neutral pH (4-5). The activity of both washed once with 0.9% NaCl, and stored at -20°; the yield of these enzymes is enhanced by GTP and by other nucleoside the various E. coli was 11-12 g of cells per 10 liters of culture phosphates in vitro (6); guanosine 5'-diphosphate-3'-diphos- medium. phate is an inhibitor of the biosynthetic ODC in vitro (7). Based Extraction of the ODC Inhibitor and of the ODC Activator on this observation, Holtta et al. (7) suggested that RNA syn- from E. coli MA255. Fractionations and enzyme manipulations thesis and polyamine synthesis are coordinately regulated in were performed at 10-40; pH measurements were performed vivo by the intracellular level of these nucleotides. Sakai and at room temperature. We have found the E. coli ODC to be Cohen (8) tested this hypothesis in a K+-requiring strain of E. particularly sensitive to inhibition by S042- therefore, every coli and reported that there was little, if any, correspondence attempt was made to ensure their removal. between the intracellular levels of putrescine and the intra- The ODC inhibitor was extracted by suspending 100 g of The publication costs of this article were defrayed in part by page Abbreviations: ODC, ornithine decarboxylase; ppGpp, guanosine 5'- charge payment. This article must therefore be hereby marked "ad- diphosphate-3'-diphosphate; PLP, pyridoxal phosphate; speA, arginine vertisement" in accordance with 18 U. S. C. §1734 solely to indicate decarboxylase; speB, agmatine ureohydrolase; speC, ornithine de- this fact. carboxylase; argE, acetyl ornithinase. 4699 4700 Biochemistry: Kyriakidis et at. Proc. Nati. Acad. Sci. USA 75 (1978) Table 1. Partial purification* of ODC from E. coli AB 1203 ODC Specific Yield of activity, activity, activity, Fraction Protein, mg units units/mg % I, supernatant 644 4996 7.7 100 II, (NH4)2SO4 134 1557 11.6 31 III, G-200 (1st) 20 1167 134 23 IV, G-200 (2nd) 3.2 945 292 19 * Calculated from the values obtained with the undiluted samples; see Fig. 1. MA255 cells in 400 ml of assay buffer A (50 mM Tris-HCl, pH The supernatant fluid contained the ODC activator; it was di- 8.2/0.1 mM EDTA/50,uM pyridoxal phosphate (PLP)/2.5 mM alyzed and lyophilized as described above. The ODC activator dithiothreitol. Portions of the cell suspension (100 ml) were has been purified approximately 100-fold by Sephadex G-75 sonicated for 12 min in a -10° bath and centrifuged at 10,000 column chromatography. A similar activating nondialyzable X g for 10 min. Solid ammonium sulfate was added to the re- factor has been partially purified from the parent ODC+ strain sultant supernatant fluid, to 100% saturation; the suspension MA197. was stirred for 20 min and then centrifuged at 10,000 X g for Assay for ODC, ODC Inhibitor, and ODC Activator. En- 10 min, and the supernatant fluid was collected. The pellet was zyme assays were performed as described (11, 14). The reaction resuspended in saturated ammonium sulfate solution (pH 7.6) mixture was modified to conform to the optimal requirements and the extraction was repeated seven times. The supernatant of E. coli ODC and contained in 0.05 ml: 2.5 ,umol of Tris-HCl fluids from each extraction were exhaustively dialyzed against buffer, pH 8.2; 0.05 jmol of EDTA; 0.002 ,umol of PLP; 0.12 buffer B (1 mM Tris-HCl, pH 8.2/0.002 mM EDTA/0.02 mM ,umol of dithiothreitol; 0.028 ,umol of DL-[1-14C]ornithine. To dithiothreitol) for 2-3 days, with three buffer changes per 24 the assay mixture, 1-2 units of purified ODC activity was added hr, until no S042- was detectable and then lyophilized. More (1 unit of enzyme activity is defined as 1 nmol of CO2 released than 80% of the total inhibitory activity was recovered in the per hour). One unit of inhibitor or activator is defined as the first four extractions. Further purification of the E. coil inhibitor amount of inhibitor or activator that inhibits or activates ODC was performed by sequential chromatography on Sephadex by 1 unit of enzyme activity, respectively, as determined in the G-75 and G-50 columns to yield a final purification factor of region of linear enzymatic response. 2000 with 80% recovery. A similar inhibitory molecule has been Purification of ODC. Ornithine decarboxylase was partially extracted from the parent ODC+ strain MA197. purified from E. coli AB1203 (ArgE) and from K-12 with The ODC activator was extracted by first suspending the modifications of the procedures described by Holtta et al. (6) saturated ammonium sulfate pellet in 60% saturated ammo- and Applebaum et al. (5). Approximately 20-30 g of cells was nium sulfate and centrifuging the suspension at 10,000 X g for sonicated in buffer A (10 g/100 ml) and centrifuged at 100,000 10 min. The resultant pellet was suspended in 40% saturated X g for 1 hr. To the supernatant fraction (fraction I), solid ammonium sulfate and centrifuged at 10,000 X g for 10 min. ammonium sulfate was added and the pellet precipitating be-

7 0

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._ V

'a4

0cn :' 3 5322 -0 *0

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a '1128YG4_1/32V6 l/8 1/4 1/2 - 1 Fractional dilution of each primary sample FIG. 1. Portions from each fraction in the purification scheme were diluted with assay buffer to provide an initial ODC activity of approximately 40,000 cpm. These fractions were then diluted as indicated. 0, Fraction I; X, fraction II; *, fraction III; o, fraction IV. Biochemistry: Kyriakidis et al. Proc. Natl. Acad. Sci. USA 75 (1978) 4701 tween 35% and 50% saturation was isolated (fraction II). This was suspended in approximately 10% of the original volume of buffer C (10 mM Tris-HCl/50 mM KCl/1 mM EDTA/0.05 mM PLP/1 mM dithiothreitol, pH 7.4), dialyzed overnight CA I against 6 liters of buffer C, applied to a Sephadex G-200 column E c (2.6 cm X 100 cm equilibrated with buffer C), and eluted with CU 0 co buffer C. The active fractions were pooled (fraction III), con- C._ CN centrated by ultrafiltration (Amicon PM 10 filter), and reap- , a plied to the same Sephadex G-200 column. After re-elution with 0 0S buffer C, the enzymatically active fractions were pooled C. 0 (fraction IV), concentrated by ultrafiltration, and stored at L- 0 -70°. .0 RESULTS Effect of Dilution on ODC Activity. A typical partial pu- 0 35 70 105 140 175 210 rification of ODC isolated from E. coli AB1203 is summarized Elution volume, ml in Table 1. The recovery of ODC activity after the first three FIG. 2. Chromatography of the dialyzed extract of E. coli MA255 (speB, speC) on Sephadex G-75 (2.7 X 40 cm). The activities of ODC fractionation steps was approximately 25%; this compares well activator (0) and ODC inhibitor (@) were assayed and the total units with the recovery of 10% of the ODC activity reported by for each fraction was calculated. Alb., albumin; Cyto. c, cytochrome H6ltti et al. (6). c. ODC activity was assayed after making serial dilutions of the various enzyme fractions obtained by this purification scheme. The graphic presentation of dilution factor versus the relative of these macromolecules, it was found more convenient to use ODC activity of a fraction at the stated dilution is provided in the ammonium sulfate fractionation scheme described in Fig. 1 and is calculated as follows: Materials and Methods. Inhibitor of E. coli ODC. Titration of 3 units of ODC, ODC activity of the partially purified from AB1203 (ArgE) or from K-12, with the undiluted fraction 2000-fold purified ODC inhibitor showed a biphasic activity Relative activity = X dilution factor. curve with a point of inflection at 0.75 ig of inhibitor protein ODC activity of the (Fig. 3). This ODC inhibitor of E. coli also was inhibitory to rat diluted fraction liver ODC; in this latter case, a linear inhibition was observed with a shallow slope that corresponds more closely to the less- If ODC activity were to decrease linearly with respect to sensitive inhibition curve observed upon titration of the E. coli dilution, a line parallel to the abscissa should be obtained ODC. starting at the ordinate value of 1.0. Fig. 1 shows that this does This 15,000 ± 2000 dalton ODC inhibitor was found to be not occur and the highest dilution (1:128) of the crude extract, sensitive to trypsin. However, the ODC inhibitor activity was fraction I, showed an 8-fold greater ODC activity than expected unaffected after incubation with alkaline phosphatase, RNase theoretically. Comparable dilutions of the partially purified A, RNase T1, DNase, 03-galactosidase, ,B-glucosidase, or pan- fractions II and III also displayed increases in activity, whereas creatic lipase. Heating to 100° for 3 min resulted in approxi- the last and most highly purified fraction (fraction IV) did not mately 30% loss of activity. increase in activity upon dilution. This result suggested that When E. coli ODC was titrated with ODC inhibitor and the factors exist in the crude extract that can inhibit ODC activity mixture was treated with ammonium sulfate and eluted and that these factors can be removed during purification. We through a Sephadex G-75 column, active ODC and active ODC attempted to enhance the production of such inhibitory factors inhibitor could be recovered. The experimental details are (11) by growing E. coli K-12, MA135 (speB), MA163 (speA), presented in the legend to Fig. 4. This experiment is similar to and AB1203 (ArgE) in the presence of 0.1 mM putrescine and that previously described with the eukaryote ODC and the increasing spermidine concentrations. A progressive decrease corresponding ODC-antizyme (2); it shows correspondingly in ODC activity occurred with increasing spermidine con- that the E. coli ODC inhibitor does not inactivate E. coli ODC centrations to 50 mM, resulting in a maximal 92% inhibition irreversibly by proteolytic cleavage. of ODC activity; however, even at the highest spermidine Activator of E. coli ODC. Titration of E. coli ODC by the concentration there remained residual assayable ODC activity ODC activator provided a linear increase of the ODC activity (results not shown). to a maximum of 6-7 times the starting value. This activity was The Macromolecular Inhibitor and the Activator of E. coli not affected by trypsin even after a 24-hr incubation at 250 ODC. It was not possible from these results to determine (trypsin/ODC activator, protein ratio of 10:1), nor did the ac- whether the decrease in the activity of ODC was due to the tivity decrease after heating at 1000 for 5 min. The molecular induction of a noncompetitive inhibitor of ODC, similar to the weight of this factor is well below 60,000 but has not been de- ODC antizyme (1, 2). To assay extracts directly for noncom- termined because the material tends to be delayed in Sephadex petitive inhibitors to ODC, the ODC activity of the extracts to columns. be tested must be zero (14). It was therefore necessary to use Fluctuations of Activities of ODC, ODC Activator, and strains of E. coli that lack ODC activity. E. coli strain MA255 ODC Inhibitor with Growth of E. coli. The activities of the (speB, speC mutant) lacks both ODC and agmatine ureohy- ODC activator and the ODC inhibitor per 106 cells were de- drolase activities and requires putrescine or spermidine for termined during the growth of E. coli MA255 in the presence normal growth (10). of low concentrations of putrescine required for optimal growth Chromatography of the crude sonicated extract of MA255 plus spermidine (Fig. 5). The activity of ODC per 106 cells was cells on Sephadex G-75 provided two distinct peaks of activity, determined during the growth of E. coli MA197, ODC+, the a fraction that stimulated ODC activity and a fraction that parent strain of MA255, ODC-. The activities of ODC and of inhibited ODC activity (Fig. 2). For large-scale preparations the ODC activator were maximal at the early stages of growth; 4702 Biochemistry: Kyriakidis et al. Proc. Natl. Acad. Sci. USA 75 (1978)

C 0

0 00 8,5 10 250 0,

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0 10 20 30 40 ODC inhibitor,,pg protein FIG. 3. Titration of E. coli ODC (0) and rat liver ODC (A) with the purified ODC inhibitor derived from MA255 (speB, speC). (Inset) Ti- tration of E. coli with low concentrations of ODC inhibitor. they then progressively decreased. Their most rapid rate of These results also show that the ratios of the activities of ODC decrease occurred late in the logarithmic phase of growth and to ODC activator to ODC inhibitor per bacterial cell are ap- coincided with the maximal activity of the ODC inhibitor; from proximately 100:50:1; these values are of course based on the the end of the logarithmic phase to the beginning of the sta- ODC activity of MA197 and on the activities of the ODC in- tionary phase, all three activities decreased in concert.

A 1.0. 0

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L- ._ c 0 1 2 3 4 Elution volume, ml 8 10 1 2 FIG. 4. Chromatography of ammonium sulfate-treated ODC- Growth, hr ODC inhibitor complex. (A) Controls. E. coli ODC (7.5 units) in buffer A was eluted through a Sephadex G-75 column (0.6 X 10 cm) equili- FIG. 5. Fluctuations in the activity of ODC (-) per 106 MA197 brated with buffer A; the recovery of ODC activity (0) was 85%. E. (speB) cells and of the activities of ODC activator (0) and ODC in- coli ODC (7.5 units) in buffer A was mixed with 3 units of E. coli ODC hibitor (0) per 106 MA255 (speB, speC) cells with growth of the cells. inhibitor (2000-fold purified); this mixture was eluted through the MA197 cells were grown in Luria's medium and the ODC activity was same Sephadex column; 85% of the unreacted ODC activity (Q) was determined in the cell extracts obtained by sonication after different recovered. (B) Experimental. A similar mixture of ODC-ODC in- times of growth. MA255 cells were grown in Luria's medium con- hibitor was treated with 10% saturated ammonium sulfate (15 min taining low putrescine and spermidine concentrations (1.12 and 0.68 at 250) and then eluted through the same column with buffer A; the mM, respectively) that were shown to be necessary for optimal growth recovery of ODC activity (0) was 80% of the original ODC activity; (10). At the indicated times, cells were sonicated and the levels of ODC the recovery of ODC inhibitor-(0) was 95% of the original ODC in- inhibitor and ODC activator were determined in the extracts. a, End hibitor. The ammonium sulfate appeared late in the elution of this of logarithmic phase of growth; b, beginning of stationary phase of column. growth. Biochemistry: Kyriakidis, et al. Proc. Natl. Acad. Sci. USA 75 (1978) 4703 produce further inhibition of ODC activity. The exact reason for the discontinuity in the curve is unknown; however, it could lj 30 600 be due to the interaction of two forms of ODC with the ODC antizyme or due to the interaction of two distinct forms of the C C ODC antizyme with ODC. Nevertheless, the similarity in the D 10 / 20.0. oL titration curve of rat liver ODC by the E. coli ODC inhibitor to that of the less-sentitive portion of the titration curve of the E 20 .4oE E. coli ODC suggests that these two enzymes may have C ~~~~~~~~~~C structurally similar sites. The ODC antizyme and the ODC activator clearly have the potential to provide both positive and negative post-translational T3 10 20> modulation of ODC activity in vivo; this is especially likely because both the inhibitory and activating macromolecules have been identified in the parent ODC+ strain MA197, from which they can be extracted and partially purified. To the ex- 0 1 2 3 4 5 6 tent that can be determined at this level of purification, these Putrescine + spermidine, mM appear to have the same general properties as the comparable until the FIG. 6. Variation in the total activities of E. coli ODC inhibitor macromolecules derived from MA255. Nevertheless, and E. coli ODC activator extractable from E. coli MA255 (speB, molecular interactions among the ODC antizyme, the ODC speC) with increasing putrescine and spermidine concentrations. activator, and ODC can be detailed, the mechanism of modulation of ODC activity will remain a matter of speculation. hibitor and of the ODC activator of MA255 cells in the absence of ODC activity. However, it is too early to derive conclusions We express our deep appreciation to Dr. Barbara J. Bachmann, about the relative contributions of the ODC activator and of Department of Human Genetics, Yale University, for the generous in supplies of the E. coli strains and to Dr. K. Brooks Low, Department the ODC inhibitor to the final expression of ODC activity of Radiobiology, Yale University, for his assistance and helpful dis- the cell. cussions. This work was supported in part by Research Grants BC260 Effect of Polyamines on the Levels of ODC Inhibitor and from the American Cancer Society and CA 04823 from the U.S. Public ODC Activator. Fig. 6 represents the changes in the specific Health Service; E.S.C. is a U.S. Public Health Service Research Career activities of the ODC inhibitor and ODC activator when the Professor (5 K06-GM-03070). MA255 cells were grown for 3.5 hr in the presence of increasing amounts of putrescine and spermidine. The specific activity of the ODC inhibitor increased approximately 20-fold when 1. Fong, W. F., Heller, J. S. & Canellakis,E. S. (1976) Biochim. Biophys. Acta 428,456-465. the total level of these two polyamines was progressively in- 2. Heller, J. S., Fong, W. F. & Canellakis, E. S. (1976) Proc. Natl. creased to 4.5 mM, whereas the specific activity of the ODC Acad. Sci. USA 73,1858-1862. activator increased maximally only by 1.6-fold. This results in 3. Canellakis, E. S., Heller, J. S., Kyriakidis, D. A. & Chen, K. Y. a final ratio of ODC activator units to ODC inhibitor units of (1978) in Advances in Polyamine Research, eds. Campbell, R. approximately 3:1 or 4:1 in contrast to the ratio of 50:1 when A., Morris, D. R., Bartos, D., Daves, G. D. & Bartos, F. (Raven, these cells are grown in the absence of polyamines. New York), Vol. 1, pp. 17-30. 4. Morris, D. R. & Pardee, A. B. (1966) J. Biol Chem. 241, 3135- DISCUSSION 3138. 5. Applebaum, D. M., Sabo, D. L., Fisher, E. H. & Morris, D. R. The response of the intracellular activity of the E. coli ODC (1975) Biochemistry 14,3675-3681. inhibitor to increases in the polyamine concentration in the 6. H6ltti, E., Jinne, J. & Pispa, J. (1972) Biochem. Biophys. Res. medium is similar to that of the ODC antizyme of eukaryote Commun. 47,1165-1171. cells when these are exposed to high polyamine concentrations. 7. H6ltta, E., Jinne, J. & Pispa, J. (1974) Biochem. Biophys. Res. Commun. 59, 1104-1111. In accord with the previously proposed terminology, this 8. Sakai, T. T. & Cohen, S. S. (1976) Proc. Natl. Acad. Sci. USA 73, macromolecule should also be termed "ODC antizyme." The 3502-3505. ratio of ODC activator to ODC antizyme varies from 50:1 to 9. Hafner, E. W., Tabor, C. W. & Tabor, H. (1977) J. Bacteriol. 132, 3:1 or 4:1 depending on the stage of growth of the cells and the 832-840. polyamine concentration of the medium. The exact mode of 10. Cunningham-Rundles, S. & Maas, W. K. (1975) J. Bacterol. 124, interaction of these effector molecules with ODC will become 791-799. clarified only when they have been highly purified. It may then 11. Heller, J. S., Chen, K. Y., Kyriakidis, D. A., Fong, W. F. & Can- also become possible to define the intrinsic activity of ODC ellakis, E. S. (1978) J. Cell Physiol. 96, 225-234. 12. Vogel, H. S. & Bonner, D. M. (1956) J. Biol. Chem. 218, 97- unaffected by its association with ODC activator and ODC 106. antizyme. 13. Ames, B. N., Garry, B. & Herzenberg, L. A. (1960) J. Gen. Mi- Titration of partially purified E. coli ODC with ODC an- crobiol. 22, 369-378. tizyme generates a curve with a sharp break; beyond this point, 14. Heller, J. S., Kyriakidis, D. A., Fong, W. F. & Canellakis, E. S. much larger additions of ODC antizyme become necessary to (1977) Eur. J. Biochem. 81,545-550.