Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

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.     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.     

Endogenous levels of polyamines and abscisic acid in pepper fruits during growth and ripening

The levels of polyamines (PA) and abscisic acid (ABA) in the pericarp of California variety pepper fruit ( Capsicum annuum L.) were analyzed during development and ripening. Putrescine level was 2.75 μmol g⁻¹ fresh weight 7 days after fruit set and fell during the exponential stage of growth to 1.05 μmol g⁻¹ fresh weight. During the second growth stage. PA and ABA levels remained stable and fell sharply at the beginning of maturation. The levels of spermidine and spermine decreased throughout fruit development and maturation from 0.61 to 0.05 and 0.31 to 0.02 μmol g⁻¹ fresh weight, respectively, but no changes were associated with the onset of maturation. ABA levels remained high (0.70-0.80 μg g⁻¹ fresh weight) during the stages of fruit growth and fell at the beginning of maturation to 0.12 μg g⁻¹ fresh weight, before rising again during the last stages of maturation and senescence. The decrease in putrescine and ABA levels and the subsequent increase in the latter may be responsible for controlling the processes of ripening in pepper fruit.     

Metabolism of l[U-14C]-arginine and l[U-14C]-ornithine in maturing and vernalised embryos and megagametophytes of Picea abies

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these 14 C-labelled amino acids into liberated CO₂, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.     

Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

In vitro interactions between polyamines and pectic substances

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.     

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.     

Inhibition of electron transport activities in mitochondria from avocado and pepper fruit by naturally occurring polyamines

The effects of the naturally occurring polyamines, spermine, putrescine, and spermidine were explored on mitochondrial state 3. state 4, and uncoupled respiration activities, ADP/O ratio, respiratory control ratio of pepper ( Capsicum annuum L. cv. Early Cal Wonder) and avocado ( Persea americana Mill. cv. Booth-8 or Simmonds) mitochondria oxidizing either succinate, external NADH, malate, α-ketoglutarate or tetramethyl- p -phenylenediamine. Abnormally high concentrations of spermine and spermidine such as might occur during chilling stress of these chilling-sensitive fruits were detrimental to several oxidase activities, especially to external NADH oxidase. State 3 respiration for NADH oxidase was inhibited more than 70% by 10 m M spermine. The spermine inhibition of uncoupled NADH oxidase was not reversed by the presence of divalent cations including Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ at concentrations up to 10 m M or by 100 m M KCl. The inhibition primarily affected the V_max. Other possible sites of polyamine interactions are discussed.     

Polyamines in Rhizobium, Bradyrhizobium, Azorhizobium and Argobacterium

Abstract Eighteen strains of Rhizobium including four species, R. leguminosarum, R. meliloti, R. loti and R. fredii , nine strains of Bradyrhizobium japonicum and three strains of Azorhizobium caulinodans contained putrescine and honospermidine as major polyamines. All these nodulating N₂-fixing rhizobia lack spermidine. Spermidine and cadaverine were present only in a limited number of R. meliloti and B. japonicum . Polymanine-synthetic activity was not affected by the differences in ability to produce phytoxine (rhizobitoxine and dihydrorhizobitoxine) H₂-uptake-hydrogenation in the organisms. Putrescine and homospermidine were major polyamined in a strain of Agrobacterium rhizogenes . All the eight strains of Agrobacterium tumefaciens as well as A. rubi, A. radiobacter and two other strains of A. rhizogenes contained putrescine and spermidine as major polyamines and homospermidine and spermine (and thermospermine) as minor polyamines.     

Enzymatic Synthesis of a Branched Trisaccharide, 2,4-di-α-Glucosyl-glucose

New procedures for determining putrescine, spermidine and spermine were first established here by the end point assay method using polyamine oxidase from Penicillium chrysogenum or Aspergillus terreus and putrescine oxidase from Micrococcus rubens. Method 1: Spermidine and spermine were first oxidized with polyamine oxidase (step A). To the reaction mixture, putrescine oxidase was added to oxidize putrescine (step B). Putrescine and spermidine in another reaction mixture were oxidized with putrescine oxidase (step C). Method 2 : Putrescine and spermidine were first oxidized with putrescine oxidase (step A). To the reaction mixture, polyamine oxidase was added to oxidize spermine (step B). Spermidine and spermine in another reaction mixture were oxidized with polyamine oxidase (step C). The amounts of putrescine, spermidine and spermine were determined from the absorbance values at each steps A, B and C.     

Inhibition of Na,K-ATPase from chick brain by polyamines.

The amounts of the polyamines putrescine, spermine and spermidine as well as the Na,K-ATPase activity have been determined in the developing chick brain. The amounts of spermine and spermidine per gram fresh weight do not change significantly, the amount of putrescine declines until the 17th day of incubation after which an increase takes place. Spermine is able to inhibit the Na,K-ATPase from chick brain competitively. Half maximal inhibition is achieved at 4 X 10(-5) mol/1 spermine. This polyamine functions as an allosteric inhibitor; the Hill coefficient is 2.2 +/- 0.3. A regulatory effect of spermine on the Na,K-ATPase from chick brain is discussed. In contrast to spermine 1 mmol/1 spermidine inhibits the Na,K-ATPase only slightly, while 1 mmol/1 putrescine does not inhibit the Na,K-ATPase at all.     

Changes in polyamine concentration during seed germination

Phaseolus mungo seeds 0 to 10 days after germination contained putrescine, spermidine, spermine, cadaverine, agmatine and tyramine. The rate of biosynthesis of total polyamines, proteins and RNA in the developing seeds follows similar profiles, reaching maxima 3 hr from germination. Putrescine, cadaverine, spermidine, spermine and agmatine were the major amines found in Pisum sativum 0–7 days after germination. RNA and proteins seem to follow the same pattern as polyamines during the first 12 hr in the developing pea seeds. RNA reaches a peak at 15 hr and polyamines and proteins peak 24 hr after germination. A rise to total polyamine concentration was also observed in seeds of Tragopogon porrifolius, Zea mays and Triticum aestivum 2–12 hr after germination.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or α-difluoromethylornithine

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and α-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G₁ and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 10⁵), medium (5 × 10⁵), or high (2 × 10⁶) cell densities in a 25 cm² flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

Occurrence of spermine in chromatin of Zea mays

Chromatin prepared from maize shoot tips using as extraction medium including quinacrine as an inhibitor of polyamine oxidase, contained 1.6 pmol spermidine g DNA^-1 and 14.8 pmol spermine g DNA^-1, respectively. This represented 0.1% spermidine and 3.7% spermine as compared with the content of those amines in the whole tissue. No putrescine was detectable in the chromatin preparation. When contamination of polyamines in the preparation was determined by the addition of labeled polyamines to the extraction medium, the ratio of the polyamines in the preparation to those in the extraction medium was 0.1% spermidine and 0.7% spermine, respectively. Spermine in the chromatin preparation was almost fully solubilized by a DNase-treatment, but spermidine was less easily solubilized. Most of the spermine associated with the chromation is chromatin-specific.     

Blood brain barrier breakdown in brain edema following cold injury is mediated by microvascular polyamines

A focal freeze injury to rat cerebral cortex induces an early (less than 5 min) increase in brain ornithine decarboxylase activity and an accumulation of polyamines involving cerebral microvessels. This polyamine synthesis correlates with the abnormal increase in microvascular permeability, monitored by uptake of Evans Blue and sod. fluorescein. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine suppressed the injury-induced increment in spermidine and spermine and microvascular permeability. Putrescine nullified alpha-difluoromethylornithine inhibition and restored microvessel spermidine and spermine and the pathological increase in microvascular permeability. These results indicate that polyamine synthesis is obligatory for blood-brain barrier breakdown. alpha-Difluoromethylornithine may be useful in the treatment of vasogenic brain edema.     

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica

Abstract: The metabolism of polyamines was investigated by injecting purified [³H]putrescine directly into the soma of the giant neuron R2 of Aplysia . Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [³H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10-⁶ M , metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10⁻⁴ M , relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S -adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of ³H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.