A luminescence-based test for determining ornithine decarboxylase activity

A sensitive chemiluminescence-based method for the assay of ornithine decarboxylase (ODC) has been developed. This method, which permits the detection of putrescine (the product of ODC) at a picomolar range, can be used to determine ODC activity in cellular extracts. Extracts are incubated with ornithine and spotted onto p81 phosphocellulose paper strips. After drying, the papers are washed with ammonium hydroxide to remove contaminants, which may interfere with the assay. Putrescine is next eluted from the paper by shaking in an elution buffer containing magnesium sulfate. Partially purified hog kidney diamine oxidase is then used to oxidize putrescine in the eluate. The hydrogen peroxide formed during the oxidation is determined by chemiluminescence using luminol and peroxidase. This simple analytical method has the sensitivity of conventional assays based on the use of radioactive ornithine.     

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.     

Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

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.     

Relationships between polyamine metabolism and RNA synthesis in post-ischemic liver cell repair

In liver cells recovering from reversible ischemia the increase in RNA synthesis by isolated nuclei is preceded by activation of ornithine decarboxylase, leading in turn to an increase in putrescine concentration. Treatment of the animals with 1,3-diaminopropane and putrescine prevents ornithine decarboxylase activation but does not hinder the enhancement of RNA synthesis in post-ischemic liver nuclei; therefore, ornithine decarboxylase activation does not seem to be a necessary prerequisite for the increase in RNA synthesis. Hypophysectomy does not prevent the post-ischemic increases of ornithine decarboxylase and RNA synthesis; but pre-treatment of the animals with cycloheximide—which has a dual effect on the activity of ornithine decarboxylase—abolishes the post-ischemic enhancement of RNA synthesis. In contrast with regenerating liver, changes in ornithine decarboxylase activity and putrescine concentrations in reversible ischemia are not associated to changes in S-adenosylmethionine decarboxylase activity and in spermine and spermidine concentrations that seem to be characteristic of tissues where increases in RNA synthesis are followed by DNA synthesis and cell multiplication.     

Regulation of polyamine synthesis in physarum polycephalum during growth and differentiation

The levels and synthesis of polyamines were investigated in Physarum polycephalum to obtain information about their regulation during growth and differentiation in a lower eukaryote. Putrescine pools rapidly increased 4–5 fold during the change from dormant spherules to growing plasmodia. The activity of ornithine decarboxylase (EC 4.1.1.17), which converts ornithine to putrescine, reflected this rapid change in the level of putrescine. Spermidine levels were closely correlated with protein concentrations during differentiation due to variations in the activity of S-adenosyl-l-methionine decarboxylase which is involved in the conversion of putrescine to spermidine This enzyme was not stimulated by putrescine, unlike the similar enzyme in other eukaryotes, thereby permitting independent regulation of putrescine and spermidine levels. The high levels of both putrescine and spermidine suggest separate functions for these polyamines in Physarum.The half-lives of ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase were 14 and 21.5 min, respectively. These short half-lives keep the polyamine metabolism under a very tight control as illustrated by the rapid fluctuations in enzyme activity during differentiation and the synchronous mitotic cycle. The step patterns of these unstable enzymes during the mitotic cycle suggest that these enzyme levels are limited by gene dosage.     

Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Effects of external osmolality on polyamine metabolism in HeLa cells

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation.A sudden increase in NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Effects of external osmolality on polyamine metabolism in HeLa cells.

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation. A sudden increase in a NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Inhibition of ornithine decarboxylase activity and spermidine accumulation in regenerating rat liver.

Injections of 1,3-diaminopropane, a close structural analogue of putrescine (1,4-diaminobutane), into partially hepatectomized rats powerfully inhibited ornithine decarboxylase (EC 4.1.1.17) activity in the regenerating liver in vivo. The compound did not have any effect on the enzyme activity in vitro (under assay conditions employed) but appeared to exert an inhibitory influence on the synthesis of ornithine decarboxylase itself.Repeated injections of diaminopropane into rats after partial hepatectomy, starting at the time of the operation and continued until 33 h postoperatively, markedly diminished the stimulation of ornithine decarboxylase activity in the regenerating liver remnant, and completely prevented the increases in hepatic spermidine concentration normally occurring in response to partial hepatectomy.Treatment of the rats with diaminopropane did not depress the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) in the regenerating liver. Nor did the compound have any effect, whatsoever, on the activity of spermidine synthase (EC 2.5.1.16) in vitro, thus obiviously proving that the increased accumulation of liver spermidine after partial hepatectomy primarily depends upon a stimulation of ornithine decarboxylase activity and a concomitant accumulation of putrescine. The results also showed that 1,3-diamino-propane could not replace putrescine in the synthesis of higher polyamines in rat liver. The inhibition of ornithine decarboxylase by diaminopropane thus appears to represent “gratuitous” repression of polyamine biosynthesis and might conceivably be used for studies devoted to the elucidation of the physiological functions of natural polyamines.     

Activities of arginine and ornithine decarboxylases in various plant species

In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V_max, ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. α-Difluoromethylornithine and α-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.

No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.

In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum.

     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

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.     

Putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in Neurospora crassa

Neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. Using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. When the pathway was blocked between putrescine and spermidine, ornithine decarboxylase synthesis rose 4-5-fold, regardless of the accumulation of putrescine. This indicates that spermidine is a specific signal for the repression of enzyme synthesis. When both putrescine and spermidine synthesis were reduced, the half-life of the enzyme rapidly increased 10-fold. The presence of either putrescine or spermidine restored the normal enzyme half-life of 55 min. Tests for an ornithine decarboxylase inhibitory protein ("antizyme") were negative. The regulatory mechanisms activated by putrescine and spermidine account for most or all of the regulatory amplitude of this enzyme in N. crassa.     

GTP-insensitive ornithine decarboxylase in acetobacteria able to synthesize spermine

Several Acetobacteria contained large amounts of spermine in addition to the putrescine and spermidine, which are the polyamines normally found in prokaryotes. A spermine synthase present in cell extracts of these Acetobacteria is the first example of this enzyme in prokaryotes. Dicyclohexylammonium sulphate inhibited both spermidine synthase and spermine synthase activities in Acetobacteria. Their ornithine decarboxylase was not stimulated by GTP nor inhibited by ppGpp and pppGpp (magic spots I and II) in contrast to ornithine decarboxylase of nearly all bacteria studied so far. However, their S-adenosyl-L-methionine decarboxylase resembled other prokaryotic adenosylmethionine decarboxylases in requiring Mg2+ ions in vitro for full activity.     

Adjustment of polyamine contents in Escherichia coli.

Adjustment of polyamine contents in Escherichia coli was studied with strains of Escherichia coli producing normal (DR112) and excessive amounts of ornithine decarboxylase [DR112(pODC)] or S-adenosylmethionine decarboxylase [DR112(pSAMDC)]. Although DR112(pODC) produced approximately 70 times more ornithine decarboxylase than DR112 did, the amounts of polyamines in the cells of both strains did not change significantly. The amounts of polyamines in DR112(pODC) were adjusted by excretion of excessive amounts of putrescine to the medium. When ornithine was deficient in cells, polyamine contents in DR112(pODC) were much higher than those in DR112, although polyamine contents were low in both strains. This indicates that large amounts of ornithine decarboxylase increased the utilization of ornithine for putrescine synthesis. During ornithine deficiency, strain DR112 produced 3.4 times more ornithine decarboxylase. Strain DR112(pSAMDC) produced seven times more S-adenosylmethionine decarboxylase than DR112 did. In DR112(pSAMDC) an increase (40%) in spermidine content, a decrease (35%) in putrescine content, and no significant excretion of putrescine and spermidine were observed. The amount of ornithine decarboxylase in DR112(pSAMDC) was approximately 30% less than that in DR112. In addition, S-adenosylmethionine decarboxylase activity was strongly inhibited by spermidine. A possible regulatory mechanism to maintain polyamine contents in Escherichia coli is discussed based on the results.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.     

Amine-specificity of the inactivating ornithine decarboxylase modification in Physarum polycephalum.

The enzyme catalysing the polyamine-stimulated modification of Physarum ornithine decarboxylase in vivo was partially purified and its activity on purified ornithine decarboxylase was examined with respect to its specificity for various amines. Spermidine, spermine and several polyamine analogues strongly promoted this reaction in vitro (apparent Km in the 0.1--0.5 mM range), whereas putrescine (apparent Km 5.33 mM) and several related diamines were not nearly as effective. In agreement with this, sensitivity studies performed in vivo also suggested that cellular spermidine, and not putrescine, is critical in modulating ornithine decarboxylase activity by this post-translational control. Unlike putrescine, or other diamines, 1,3-diaminopropane demonstrated a functional similarity to the polyamines in stimulating this reaction. This study has demonstrated a method whereby non-physiological amines capable of depressing ornithine decarboxylase activity by this natural feedback mechanism can be readily identified for further evaluation of their potential use in the experimental and medical control of polyamine biosynthesis.