Polyamine distribution patterns within the families Aeromonadaceae,Vibrionaceae, Pasteurellaceae,and Halomonadaceae,and related genera of the gamma subclass of the Proteobacteria

Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromonadaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salinivibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously contained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxyspermidine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass.     

Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases

Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.     

Synthesis of novel polyamines in Paracoccus, Rhodobacter and Micrococcus

Abstract The Gram-negative facultative chemolithotroph, Paracoccus denitrificans contains putrescine, cadaverine, agmatine, spermidine, aminopropylcadaverine, spermine, thermospermine and aminopentylnorspermidine. This bacterium has the ability to produce norspermidine from supplemented diaminopropane. The halophile, Paracoccus halodenitrificans is devoid of any polyamines. Neither decarboxylation of ornithine, lysine or arginine, nor triamine synthetic activity from diamines was detected in this halophile. Two Gram-negative facultative photoautotrophs, Rhodobacter sphaeroides and Rhodobacter capsulatus contain putrescine, cadaverine, agmatine and spermidine and can produce norspermidine from supplemented diaminopropane. A Gram-negative eubacterium, Micrococcus cryophilus , contains histamine and homospermidine in addition to putrescine, cadaverine and spermidine. Hence, polyamine distribution patterns and polyamine biosynthetic activities were very different among the four groups of Gram-negative eubacteria examined.     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Whole-cell uptake and nuclear localization of 1,25-dihydroxycholecalciferol by breast cancer cells (T47 D) in culture

1. The specificity of rat prostatic spermidine synthase and spermine synthase with respect to the amine acceptor of the propylamine group was studied. 2. Spermidine synthase could use cadaverine (1,5-diaminopentane) instead of putrescine, but the Km for cadaverine was much greater and the rate with 1mM-cadaverine was only 10% of that with putrescine. 1,3-Diaminopropane was even less active (2% of the rate with putrescine) and no other compound tested (including longer alpha,omega-diamines, spermidine and its homologues and monoacetyl derivatives) was active. 3. Spermine synthase was equally specific. The only compounds tested that showed any activity were 1,8-diamino-octane, sym-homospermidine, sym-norspermidine and N-(3-aminopropyl)-cadaverine, which at 1mM gave rates 2, 17, 3 and 4% of the rate with spermidine respectively. 4. The formation of polyamine derivatives of cadaverine and to a very small extent of 1,3-diaminopropane was confirmed by exposing transformed mouse fibroblasts to these diamines when synthesis of putrescine was prevented by alpha-difluoromethylornithine. Under these conditions the cells accumulated significant amounts of N-(3-aminopropyl)cadaverine and NN'-bis(3-aminopropyl)cadaverine when exposed to cadaverine and small amounts of sym-norspermidine and sym-norspermine when exposed to 1,3-diaminopropane.     

A sequential injection analysis/chemiluminescent plant tissue-based biosensor system for the determination of diamine

In this paper, a new chemiluminescent plant tissue-based biosensor for diamine detection was presented by employing sequential injection analysis (SIA), which facilitates precise fluidic handling and lower consumption of sample and reagents. Pea-seedling tissue acted as the molecular recognition element and was packed in a mini-PTFE column and further incorporated in the SIA system. The analysis of diamines, such as putrescine and cadaverine, is based on an enzymatic conversion which takes place in the plant tissue column to produce hydrogen peroxide. The formed hydrogen peroxide was detected by a chemiluminescence reaction involving luminol and Co(2+). Under the optimal conditions, the linear calibration graphs were obtained within 0.2-80 microM (putrescine) and 0.5-100 microM (cadaverine). The detection limits of 0.03 and 0.06 microM were achieved for putrescine and cadaverine, respectively, along with the relative standard deviations of 2.14% and 3.08% (n=11) and a sampling frequency of 40 h(-1). The present biosensor has been used for the analysis of diamine in fish samples with an acceptable accuracy.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

Evidence for transglutaminase activity in plant tissue

An extract prepared from the apical meristematic region of etiolated pea seedlings was able to catalyze the incorporation of putrescine into trichloroacetic acid precipitable material. The enzyme was found to be soluble and followed a typical Michaelis-Menten kinetics when N-N-dimethyl casein was used as a substrate. Its activity was promoted by Ca²⁺ and inhibited by Cu²⁺ and dl-dithiothreitol. Other polyamines competed with putrescine as substrates and cadaverine was the most potent inhibitor of putrescine incorporation. Plant transglutaminase is capable of recognizing specific sites in substrates described for animal transglutaminase, like insulin, fibrinogen, pepsin, and thrombin. However, it can also use as substrates cellulase and creatine kinase which have not been described for transglutaminase from other sources.     

The occurrence, subcellular localization and partial purification of diamine acetyltransferase in the yeast Candida boidinii grown on spermidine or putrescine as sole nitrogen source

The yeast Candida boidinii when grown on spermidine, diaminopropane, putrescine or cadaverine as sole nitrogen source contains an N-acetyltransferase capable of acetylating the primary amino groups of spermine, spermidine, acetylspermidines, acetylputrescine and alpha, omega-diaminoalkanes. In the case of spermidine, the products were N1-acetylspermidine and N8-acetylspermidine in the ratio 50:45 with traces of other unidentified products. The enzyme was partially purified and the stoichiometry determined, together with apparent Km and V values for a number of substrates. The pH optimum was about 8.8 for putrescine and 9.3 for spermidine. The unstable enzyme was partially stabilized by 10% (v/v) glycerol or bovine serum albumin (5 mg/ml). The kinetic parameters were determined with putrescine as substrate and the mechanism shown to be of the sequential type. The enzyme was shown to be located in the mitochondria of C. boidinii, in contrast to mammalian N-acetyltransferases. The enzyme was found in a number of other yeast species when grown on spermidine or putrescine, but was only present in those species that had previously been found to contain polyamine oxidase. It is suggested that in C. boidinii, as in mammals, acetylation of spermidine and putrescine must precede their catabolism.     

Soybean polyamines: separation and characterization of cadaverine

Cadaverine in soybeans was separated by ion exchange chromatography from other polyamines previously identified. Identification of cadaverine was based on ion exchange separation, thin layer chromatography, paper electrophoresis, mass and nuclear magnetic resonance spectral analyses. Since the molecules of putrescine and cadaverine are so similar, separation and identification of the two components are difficult. Their R_F values on thin layer chromatography are close, although cadaverine produces a bluish purple color when sprayed with ninhydrin reagent, while putrescine forms a purple color. Separation likewise is poor by paper electrophoresis, gas chromatography, and gel filtration. The mass spectra of cadaverine and putrescine have m/e peaks at 30, 43, 45, 56, 73, 85, 102 and 30, 43, 59, 71, 88, respectively. The m/e peaks differentiate one compound from the other. Nuclear magnetic resonance spectra and their integration curves show that cadaverine contains two types of methylene protons (10 total) in 3:2 ratio while putrescine produces two types (8 total) in 1:1 ratio. Polyamines occur at levels of micrograms per gram of soybeans with spermidine present in the largest quantity.     

Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A- and B-DNA located by simulated annealing

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na+ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

Influence of diamines and polyamines on the senescence of plant suspension cultures

The diamines putrescine and cadaverine and the polyamines spermine and spermidine inhibited the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. Response was observed when the media of stationary phase cultures was adjusted to either 1 mM of cadaverine or putrescine; or 0.1 μM of either spermine or spermidine along with 2% sucrose in all cases. Senescence of the cultures was followed by microscopic examination of cell aliquots removed at 10 day intervals and treated with the vital stain, fluorescein diacetate.     

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.     

Binding Sites of the Polyamines Putrescine,Cadaverine, Spermidine and Spermine on A- and B-DNA Located by Simulated Annealing

Abstract

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na⁺ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

Polyamine degradation in foetal and adult bovine serum.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.     

Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis.

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine carbamoyltransferase, measured by molecular sieving, is 250 000. It is composed of six apparently identical subunits with a molecular weight of 39 000, as determined by cross-linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine carbamoyltransferase is a trimer of 140 000 consisting of three identical subunits with a molecular weight of 40 000. Ornithine carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerable lower efficiency than for putrescine. The kinetic mechanism of putrescine carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead-end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established. The properties of putrescine carbamoyltransferase are compared to the known properties of other carbamoyltransferases. The evolutionary implications of this comparison are discussed.     

Relationships between microbial population dynamics and putrescine and cadaverine accumulation during dry fermented sausage ripening

Aims:  To evaluate the concomitant effects of three technological variables (fermentation temperature, NaCl and glucose added to the meat batter) on diamines (cadaverine, putrescine and histamine) accumulation and microbial changes during ripening of dry fermented sausages.
Methods and Results:  The variables were modulated according to an experimental design and predictive mathematical models were obtained. The models indicated that the sausages were characterized by low histamine amount independently on the applied conditions. In contrast, putrescine and cadaverine accumulation was considerable and significantly affected by the three variables. The microbial population dynamics suggest that lactic acid bacteria (LAB) and microstaphylococci are favoured by increasing glucose concentration until 0·7 g kg⁻¹, while Enterobacteriaceae are negatively influenced by NaCl concentration and, to a lesser extent, by fermentation temperature.
Conclusions:  Data obtained showed a relationship between Enterobacteriaceae growth and cadaverine and putrescine accumulation in sausages during ripening. The conditions more favourable for LAB and microstaphylococci induced a reduced growth of Enterobacteriaceae with a consequent reduced accumulation of putrescine and cadaverine.
Significance and Impact of the Study:  The use of systematic experimental designs allows to individuate the technological conditions suitable to keep the aminogenic microflora under control, thus reducing the risk of diamines production during traditional fermented food manufacture.     

Correlations between polyamine ratios and growth patterns in seedling roots

The levels of putrescine, cadaverine, spermidine and spermine were determined in seedling roots of pea, tomato, millet and corn, as well as in corn coleoptiles and pea internodes. In all roots, putrescine content increased as elongation progressed, and the putrescine/spermine ratio closely paralleled the sigmoid growth curve up until the time of lateral root initiation. Spermidine and spermine were most abundant near the apices and declined progressively with increasing age of the cells. In the zone of differentiation of root hairs in pea roots, putrescine rose progressively with increasing age, while cadaverine declined. In both pea internodes and corn coleoptiles, the putrescine/spermidine ratio rises with increasing age and elongation. Thus, a block in the conversion of the diamine putrescine to the triamine spermidine may be an important step in the change from cell division to cell elongation.Supported by a Fellowship of the Peoples' Republic of China.Aided by grant 5-RO1-AGO2742 from the National Institutes of Health to A.W.G.     

Transport of putrescine across duodenal,jejunal and ileal brush-border membrane of chicks (Gallus domesticus)

Luminal polyamines and their absorption are essential for proliferation of the enterocytes and, therefore, nutrition, health and development of the animal. The transport systems that facilitate the uptake of putrescine were characterized in chick duodenal, jejunal and ileal brush-border membrane vesicles prepared by MgCl2 precipitation from three-week-old chicks. An inwardly-directed Na+ gradient did not stimulate putrescine uptake and, therefore, putrescine transport in chick intestine. In the duodenum, jejunum and ileum, kinetics of putrescine transport fitted a model with a single affinity component plus a non-saturable component. The affinity (Kt) for [3H]putrescine transport across the brush-border membrane increased along the length of the small intestine. A model of intermediate affinity converged to the data obtained for [3H]putrescine transport with Kt approximating 1.07 and 1.05 mM or duodenum and jejunum, respectively; and high affinity with a Kt of 0.35 mM for the ileum. The polyamines cadaverine, putrescine, spermidine and spermine strongly inhibited the uptake of [3H]putrescine into chick brush-border membrane vesicles, more so for the jejunum and ileum than the duodenum. The kinetics of cadaverine, spermidine and spermine inhibition are suggestive of competitive inhibition of putrescine transport. These uptake data indicate that a single-affinity system facilitates the intestinal transport of putrescine in the chick; and the affinity of transporter for putrescine is higher in the ileum than in the proximal sections of the small intestine. In addition, this study shows that the ileum of chicks plays an important role in regulating cellular putrescine concentration.