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

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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 ex- tracted 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 reac- tion. 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.
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