Control of ornithine decarboxylase activity in jute seeds by antizyme

The control of ornithine decarboxylase activity by antizyme was studied during early germination of jute seeds(Corchorus olitorius). When 2 mM of putrescine and spermidine were applied to the germinating medium, the enzyme activity was markedly inhibited (1.7-fold) during 16 h imbibition. This inhibition could be attributed to the formation of an inhibitory protein termed antizyme. The antizyme was partially purified from jute and barley seedlings. The activity of jute ornithine decarboxylase antizyme was weaker than that of barley.     

The existence of ornithine decarboxylase-antizyme complex in germinated barley seeds

Ornithine decarboxylase (ODC; EC 4.1.1.17) and its antizyme (Az), a protein non-competitive inhibitor of ODC, form a complex in germinated barley ( Hordeum vulgare L. cv. Georgia) seeds. The ODC-Az complex is very stable, but dissociates by treatment with 10% ammonium sulfate. ODC-Az complex is present in the cytosol, and it can also be extracted from germinated barley seed chromatin with 2 M NaCl.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

The dual action of the non-physiological diamines 1,3 diaminopropane and cadaverine on ornithine decarboxylase of HTC cells

In rat hepatoma tumor (HTC) cells 1,3 diaminopropane and cadaverine induced the ornithine decarboxylase antizyme as well as the end product of the ornithine decarboxylase reaction putrescine. Although at equal exogenous concentrations (10^?3M) the two non-physiological diamines penetrated the cells as effectively as putrescine; they decreased cellular ornithine decarboxylase considerably less rapidly than the naturally present diamine. Cell extracts treated with high concentrations of 1,3 diaminopropane and putrescine, and which as a result had a high specific activity of ornithine decarboxylase antizyme, were chromatographed on a superfine Sephadex G-75 column in the presence of 250 mM NaCl. No ornithine decarboxylase-antizyme complex could be detected indicating the original decrease of ornithine decarboxylase in the cells was likely due to some mechanism other than antizyme. These results indicate that 1,3 diaminopropane and cadaverine probably can act on ornithine decarboxylase, like putrescine, by two distinct regulatory mechanisms.     

Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver

A protein inhibiting a protein inhibitor (antizyme) to ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC), antizyme inhibitor, was purified from the liver cytosol of thioacetamide-treated rats by procedures including antizyme affinity chromatography. Overall purification was roughly estimated to be about 17,000,000-fold and recovery was about 2.4%. The purified preparation showed one major protein band and a faint band corresponding in mobility to molecular weights of 51,000 and 53,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Judging from the ornithine decarboxylase activity of the final preparation, the faint band may be ornithine decarboxylase. The apparent molecular weight of antizyme inhibitor estimated by gel filtration on Sephacryl S-200 was approx. 62,000, indicating that antizyme inhibitor may be composed of a single polypeptide chain. In order to examine the question of whether antizyme inhibitor is a protein derived from ornithine decarboxylase, an inactive ornithine decarboxylase, in an immunotitration study and analysis of the binding to antizyme were investigated. The results indicate that antizyme inhibitor may be a protein distinct from ornithine decarboxylase.     

A monoclonal antibody to rat liver ornithine decarboxylase

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.     

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.     

Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4–6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5μm-spermine or -spermidine or 10μm-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.     

The velocity of the reaction between ornithine decarboxylase and antizyme highly depends on the presence of salt and temperature

Antizyme reversibly inhibits ornithine decarboxylase activity by direct binding to the enzyme. The velocity of the reaction between ornithine decarboxylase and antizyme was markedly accelerated as the concentration of sodium chloride in the medium was increased and as the temperature of incubation was lowered. The equilibrium constant (binding constant) of the reaction between ornithine decarboxylase and antizyme was a little increased by decreasing salt concentrations in the medium and by decreasing the temperature of incubation.     

Analyses of ornithine decarboxylase antizyme mRNA with a cDNA cloned from rat liver

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.     

Polyamine-independent Expression of Caenorhabditis elegans Antizyme

Degradation of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis, is promoted by the protein antizyme. Expression of antizyme is positively regulated by rising polyamine concentrations that induce a +1 translational frameshift required for production of the full-length protein. Antizyme itself is negatively regulated by the antizyme inhibitor. In our study, the regulation of Caenorhabditis elegans antizyme was investigated, and the antizyme inhibitor was identified. By applying a novel GFP-based method to monitor antizyme frameshifting in vivo, we show that the induction of translational frameshifting also occurs under stressful conditions. Interestingly, during starvation, the initiation of frameshifting was independent of polyamine concentrations. Because frameshifting was also prevalent in a polyamine auxotroph double mutant, a polyamine-independent regulation of antizyme frameshifting is suggested. Polyamine-independent induction of antizyme expression was found to be negatively regulated by the peptide transporter PEPT-1, as well as the target of rapamycin, but not by the daf-2 insulin signaling pathway. Stress-dependent expression of C. elegans antizyme occurred morely slowly than expression in response to increased polyamine levels, pointing to a more general reaction to unfavorable conditions and a diversion away from proliferation and reproduction toward conservation of energy. Interestingly, antizyme expression was found to drastically increase in aging individuals in a postreproductive manner. Although knockdown of antizyme did not affect the lifespan of C. elegans, knockdown of the antizyme inhibitor led to a significant reduction in lifespan. This is most likely caused by an increase in antizyme-mediated degradation of ornithine decarboxylase-1 and a resulting reduction in cellular polyamine levels.     

Nonspecific inhibition of Escherichia coli ornithine decarboxylase by various ribosomal proteins: detection of a new ribosomal protein possessing strong antizyme activity

Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was found to be inhibited by several basic proteins. When ribosomal proteins were tested, major ribosomal proteins, with the exceptions of S1, S5, S6, S8, S10, L3, L5, L6, L7/L12, L8, L9 and L10 proteins, showed antizyme activity in addition to the recognized antizymes (S20/L26 and L34 proteins). Furthermore, it was found that L20 protein and a new ribosomal protein, tentatively named X1 protein and bound to 50 S ribosomal subunits, showed stronger antizyme activity than S20/L26 and L34 proteins. The antizyme activity of S20/L26 and L34 proteins was at most 10% of the total antizyme activity of ribosomal proteins. Several basic polypeptides also showed antizyme activity in the order polyarginine greater than protamine greater than histone greater than polylysine. Ribosomal proteins and basic polypeptides inhibited ornithine decarboxylase activity competitively. Ribosome-bound antizymes were inactive as antizymes, and antizyme inhibition of ornithine decarboxylase was eliminated by ribosomes. When E. coli extracts were separated into ribosomes and 100,000 X g supernatant fraction, no significant antizyme activity was observed in the supernatant fraction. Results of these in vitro experiments infer that basic antizymes may not function as inhibitors of ornithine decarboxylase in vivo.     

Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase.

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.     

Detection of Ornithine Decarboxylase Antizyme in Mouse Brain

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to 1,3-diamino-2-propanol, an antizyme-inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14-fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3-diamino-2-propanol about fivefold.     

Properties of ornithine decarboxylase-antizyme from mammary gland of lactating rats

Ornithine decarboxylase-antizyme was induced in mammary gland of fasted lactating rats by administration of 1,3-diaminopropan-2-ol. Antizyme from mammary gland showed similar chemical and kinetic behavior to that previously reported by Canellakis and co-workers for antizyme from liver [J. S. Heller, W. F. Fong, and E. S. Canellakis (1976) Proc. Natl. Acad. Sci. USA 72, 1858-1862]; specifically the inhibitor was nondialyzable, heat labile, and ribonuclease insensitive, and the inhibition was time independent, proportional to the concentration of antizyme present, and noncompetitive with respect to the substrate, ornithine. However, ornithine decarboxylase-antizyme from mammary gland eluted from Sephadex G-75 with an apparent molecular mass of 55 kDa, compared with 27 kDa, for antizyme from liver under identical conditions. The elution pattern was unaffected by the presence of high salt concentrations, indicating that the larger size was not due to macromolecular complexes. The presence of antizyme-ornithine decarboxylase complex was detected in mammary gland of untreated lactating rats fasted for 6 or 24 h, thus indicating that antizyme plays a role in the regulation of ornithine decarboxylase in mammary gland under physiological conditions.     

The effects of priming treatments using seaweed suspensions on the water sensitivity of barley (Hordeum vulgare L.) caryopses

Two seaweed suspensions, one prepared from Ascophyllum nodosum (Linnaeus) Le Jolis (ANS), another from Laminaria hyperborea (Gunn.) Foslie (LHS), were evaluated for their effects on the water sensitivity of barley (Hordeum vulgare L.) seeds. Priming in either seaweed suspension did not reduce viability. Priming in ANS was beneficial to seeds germinated in elevated water levels. The suspension of A. nodosum reduced the water sensitivity of barley seeds better than either water or polyethylene glycol (PEG) priming treatments. ANS also reduced the microbial population on the seeds by 86%. The hygroscopic properties and the antibiotic effect of the suspension of A. nodosum resulted in greater oxygen availability to the embryo, enabling more seeds to germinate under oxygen deficient conditions.     

Effect of starvation and refeeding on polyamine concentrations and ornithine decarboxylase antizyme in mammary gland of lactating rats.

Starvation caused a marked increase in putrescine content in mammary gland of lactating rats, together with a marked decrease in activity of ornithine decarboxylase and appearance of measurable ornithine decarboxylase antizyme. 2. Refeeding for 5 h caused disappearance of free antizyme and ornithine decarboxylase activity returned to the value in fed animals. Putrescine concentration remained elevated. 3. There was no significant change in nucleic acid content of mammary gland from starved rats, but spermidine and spermine contents increased significantly. 4. Refeeding for 5 h returned the spermidine content of mammary glands to 'fed' values, and significantly decreased the content of spermine, although it did not reach control values. Thus changes in polyamine content of mammary gland in starved rats are clearly dissociated from changes in either RNA content or activities of polyamine-synthetic decarboxylases. 5. Starvation caused a fall in the content of spermidine in liver, with no change in spermine content. Refeeding for 5 h returned the spermidine content to 'fed' values.     

Purification and properties of the antizymes of Escherichia coli to ornithine decarboxylase

The purification of the antizymes to ornithine decarboxylase of Escherichia coli to homogeneity is detailed. An acidic component, pI 3.8, and two basic histone-like proteins, pI above 9.5, are described. The two latter proteins constitute approximately 90% of the total antizyme activity.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

The Drosophila Gene for Antizyme Requires Ribosomal Frameshifting for Expression and Contains an Intronic Gene for snRNP Sm D3 on the Opposite Strand

Previously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5′ open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5′ ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype.