Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Metabolism of polyamines in mouse neuroblastoma cells in culture: formation of GABA and putreanine.

—Polyamine metabolism of mouse neuroblastoma cells grown in culture was studied with special reference to the synthesis of GABA from putrescine and putreanine from spermidine. This study shows that neuroblastoma cells in the presence of a complete culture medium containing calf serum readily metabolized [¹⁴C]putrescine to GABA; the rate of synthesis is similar to the rate of synthesis of spermidine from putrescine. In the absence of serum the conversion of putrescine to GABA is minimal. In the presence of serum GABA formation is completely inhibited by the diamine oxidase inhibitor aminoguanidine. GABA synthesis does not occur in the absence of cells. The GABA synthesized is not readily metabolized to succinate or homocarnosine. Mouse neuroblastoma cells metabolized [¹⁴C]ornithine to putrescine, GABA, and spermidine. Spermidine was metabolized to putrescine, putreanine and spermine.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

Polyamines in type 5 adenovirus-infected cells and virions

The incorporation of l-[¹⁴C]ornithine and [¹⁴C]putrescine into putrescine, spermidine, and spermine in type 5 adenovirus-infected KB cells was identical to that in uninfected control cells early in infection, but incorporation into putrescine stopped after 8 to 12 h and the rate of incorporation into spermidine was reduced between 12 and 20 h after infection. The amount of polyamines found associated with purified virus could neutralize a maximum of 3 to 4% of the virus DNA, but the small quantities of polyamines detected could not be distinguished from nonspecific binding of polyamines to virions. These data suggest that polyamines are probably not integral components of adenovirus particles.     

Regulation of putrescine metabolism in Ehrlich ascites carcinoma cells exposed to hypotonic medium

When exposed to hypotonic growth medium, Ehrlich ascites carcinoma cells showed a rapid stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in 4 h, followed by a rise in their putrescine content. This effect was totally abolished by addition of a slightly hypertonic concentration of sodium chloride or sucrose to the medium. The general protein synthesis was unaffected by the hypotonic treatment. The uptake of putrescine and, to a lesser extent, spermidine was enhanced, and the conversion of the radioactive putrescine into spermidine appeared partially inhibited during later stages of the hypotonic treatment. As a result, the half-life of putrescine increased from 2.8 h under isoosmotic conditions to 7.3 h in hypoosmotic medium. Both exogenous ([¹⁴C]-putrescine-derived) and endogenous ([¹⁴C]ornithine-derived) putrescine degraded at similar rates in control and hypotonic cells, yet the putrescine taken from the medium degraded preferably to nonpolyamine products, while the putrescine synthesized in the cell was converted evenly to spermidine and to other metabolites. Adenosylmethionine decarboxylase activity (EC 4.1.1.50), which provides the second precursor for spermidine and spermine synthesis, was distinctly inhibited in the hypotonic medium. Inhibition was likewise observed in spermidine synthase activity, while spermine synthase was marginally stimulated. It appears that the hypotonic treatment serves a special condition under which not only the formation of putrescine is enhanced dramatically but the cells also attempt to conserve the diamine by preventing its further metabolism to higher polyamines.     

Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1 alpha,25-dihydroxyvitamin D3-induced putrescine synthesis

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.     

Polyamine depletion induces enhanced synthesis and accumulation of cadaverine in cultured Ehrlich ascites carcinoma cells

An exposure of cultured Ehrlich ascites carcinoma cells to DL-α-difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), rapidly depleted the tumor cells of putrescine and spermidine. The decrease in the cellular concentrations of these two natural polyamines, however, was accompanied by a striking appearance of two new major amines: cadaverine and a compound tentatively identified as N-3-aminopropyl-1,5-diaminopentane (aminopropylcadaverine). When the cultures were grown in the presence of uniformly labeled [¹⁴C]lysine, tumor cells exposed to difluoromethyl ornithine converted lysine to cadaverine and aminopropyl cadaverine at strikingly enhanced rate. The difluoromethyl ornithine-induced accumulation and synthesis of cadaverine and aminopropylcadaverine were totally prevented by the presence of micromolar concentrations of spermidine (or spermine) in the culture media.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spormine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [³⁵S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Spermidine Biosynthesis During Germination and Subsequent Vegetative Growth of Bacillus megaterium Spores

Spermidine biosynthesis was extremely low early in germination of Bacillus megaterium spores and the spermidine level remained constant. Rapid synthesis began after 130 min and thereafter accounted for the increase in spermidine level which began at this time. Biosynthesis was greatly (>84%) diminished by exogenous spermine or spermidine. Arginine and ornithine were both converted efficiently into spermidine, but arginine was the more immediate precursor as shown by isotope competition studies and by the absence of ornithine decarboxylase and the presence of arginine decarboxylase. Exogenous putrescine was not incorporated into spermidine, although it was taken up rapidly and degraded.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Polyamines and their biosynthetic enzymes in Ehrlich ascites-carcinoma cells. Modification of tumour polyamine pattern by diamines.

1. Ehrlich ascites-carcinoma cells contained relatively high concentrations of spermidine and spermine, but the putrescine content of the washed cells was less than 10% of that of higher polyamines. 2. Ascites-tumour cells likewise exhibited high activities of L-ornithine decarboxylase (EC 4.1.1.17), S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (EC 2.5.1.16) and spermine synthase. 3. During the first days after the inoculation, the polyamine pattern of the ascites cells was characterized by a high molar ratio of spermidine to spermine, which markedly decreased on aging of the cells. 4. Various diamines injected into mice bearing ascites cells rapidly and powerfully decreased ornithine decarboxylase activity in the carcinoma cells, apparently through a mechanism that was not a direct inhibition of the enzyme in vitro. Cadaverine (1,5-diaminopentane) and 1,6-diaminohexane were the most potent inhibitors of ornithine decarboxylase among the amines tested. 5. Chronic treatment of the mice with diamines resulted in a virtually complete disappearance of ornithine decarboxylase activity, and after 24h a significant decline in spermidine accumulation. 6. Cadaverine appeared to be an especially suitable compound for use as an inhibitor of the synthesis of higher polyamines, at least in Ehrlich ascites cells, since this diamine also acted as a competitive inhibitor for putrescine in the spermidine synthase reaction without being incorporated into the higher polyamines.     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

The Biosynthesis of Polyamines in the Brain of Audiogenic Seizure-Susceptible and -Resistant Deermice

Abstract: The biosynthesis of polyamines was investigated in the brains of the audiogenic seizure-susceptible (SS) mutant and the wild-type, seizure-resistant (SR) deermouse Peromyscus maniculatus bairdii. For this purpose a new, rapid, and economical high pressure liquid chromatography (HPLC) procedure for the quantitation of putrescine, spermidine, and spermine was developed. Benzoyl derivatives of the polyamines, prepared from a crude brain supernatant, were ether extracted and, following removal of the ether, were separated and quantitated by HPLC. The high sensitivity of the method allows quantitation of putrescine in 50 mg and of spermidine and spermine, in as little as 2-2.5 mg, of brain tissue. No differences were found in endogenous levels of the 3 polyamines in brains of SS vs SR deermice. Using [¹⁴C]putrescine as a polyamine precursor, we found the specific radioactivity of spermidine to be lower in the SS than in the SR brains following a 1 h intraventricular (i.vt.) pulse. No such differences were noted if [3,4-¹⁴C]methionine was used as the polyamine precursor. To test whether the flux of methionine through the transmethylation pathway was also different in SS and SR deermouse brain, we administered [1-¹⁴C]methionine (i.vt.) (1 h pulse). Even though the brains of SS animals contained higher methionine and lower S-adenosyl-l -methionine (AdoMet) levels than the SR brains, the specific radioactivities of methionine and AdoMet were, respectively, lower and higher in SS compared to SR brains. The latter results are in agreement with our previous findings of an accelerated utilization of AdoMet in brains of Swiss-Webster mice following administration of the chemical convulsant l -methionine-d,l-sulfoximine (MSO). Taken together, the data suggest that the SS condition, whether genetically determined (as in the SS deermouse) or chemically elicited (as after MSO), correlates positively with higher than normal rates of conversion of methionine to brain AdoMet and leads to an enhanced rate of utilization of AdoMet via the transmethylation pathway.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Arginase from rat fibrosarcoma. Its possible role in proline,glutamate and polyamine metabolism

The presence of arginase in rat fibrosarcoma not synthesizing urea, suggested that this enzyme may have additional functions. Ornithine carbamoyl transferase, a key enzyme of the urea cycle was absent in this tissue, when compared to normal tissues, lower amount of ornithine was found in the fibrosarcoma, but this tumour contained a higher level of proline. The radioactivity present in L-[U-¹⁴C] arginine was incorporated into putrescine, spermidine, spermine, proline glutamate and glutamine suggesting that arginine was a possible precursor and that arginase may have a role in the synthesis of these metabolites.