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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.38, #2)
Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach by E. Agostinelli; G. Tempera; N. Viceconte; S. Saccoccio; V. Battaglia; S. Grancara; A. Toninello; R. Stevanato (pp. 353-368).
The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H2O2 and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H2O2 and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42°C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.
Keywords: Polyamine; Amine oxidases; Tumor cells; Multidrug resistance; Hyperthermia
Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases by Merja R. Häkkinen; Mervi T. Hyvönen; Seppo Auriola; Robert A. Casero Jr; Jouko Vepsäläinen; Alex R. Khomutov; Leena Alhonen; Tuomo A. Keinänen (pp. 369-381).
N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K m(APAO) = 10 μM, k cat(APAO) = 1.1 s−1 and K m(SMO) = 28 μM, k cat(SMO) = 0.8 s−1, respectively], metabolized BnEtSPM to EtSPD [K m(APAO) = 0.9 μM, k cat(APAO) = 1.1 s−1 and K m(SMO) = 51 μM, k cat(SMO) = 0.4 s−1, respectively], and metabolized DBSPM to BnSPD [K m(APAO) = 5.4 μM, k cat(APAO) = 2.0 s−1 and K m(SMO) = 33 μM, k cat(SMO) = 0.3 s−1, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K m(APAO) = 16 μM, k cat(APAO) = 1.5 s−1; K m(SMO) = 25 μM, k cat(SMO) = 8.2 s−1; BnSPM K m(APAO) = 6.0 μM, k cat(APAO) = 2.8 s−1; K m(SMO) = 19 μM, k cat(SMO) = 0.8 s−1, respectively]. Surprisingly, EtSPD [K m(APAO) = 37 μM, k cat(APAO) = 0.1 s−1; K m(SMO) = 48 μM, k cat(SMO) = 0.05 s−1] and BnSPD [K m(APAO) = 2.5 μM, k cat(APAO) = 3.5 s−1; K m(SMO) = 60 μM, k cat(SMO) = 0.54 s−1] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.
Keywords: Polyamines; N-alkylated polyamine analogues; Flavin-dependent amino-oxidoreductases; Spermine oxidase; Polyamine oxidase
Synthetic polyamines: an overview of their multiple biological activities by Anna Minarini; Andrea Milelli; Vincenzo Tumiatti; Michela Rosini; Maria Laura Bolognesi; Carlo Melchiorre (pp. 383-392).
The binding of polyamines to a variety of receptors and other defined recognition sites has been widely reported. It is well-known that polyamines interact with aspartate, glutamate, and aromatic residues of a given receptor and/or enzyme mainly through the formation of ion bonds, since at physiological pH, protonation of amino groups is nearly complete. From this, the hypothesis arises that a polyamine may be a universal template able to recognize different receptor systems. This hypothesis suggests that both affinity and selectivity may be fine-tuned by inserting appropriate substituents onto the amine functions and by varying the methylene chain lengths between them on the polyamine backbone. In this paper, we detail several application of this design strategy aimed at discovering potent and selective polyamines able to bind neurotransmitter receptors and enzymes, such as muscarinic receptor subtypes, muscle-type nicotinic receptors and acethylcholinesterase.
Keywords: Polyamines; Universal template; Methoctramine; MTDLs; Memoquin
Polyamines: fundamental characters in chemistry and biology by E. Agostinelli; M. P. M. Marques; R. Calheiros; F. P. S. C. Gil; G. Tempera; N. Viceconte; V. Battaglia; S. Grancara; A. Toninello (pp. 393-403).
Polyamines are small cationic molecules required for cellular proliferation and are detected at higher concentrations in most tumour tissues, compared to normal tissues. Agmatine (AGM), a biogenic amine, is able to arrest proliferation in cell lines by depleting intracellular polyamine levels. It enters mammalian cells via the polyamine transport system. Agmatine is able to induce oxidative stress in mitochondria at low concentrations (10 or 100 μM), while at higher concentrations (e.g. 1–2 mM) it does not affect mitochondrial respiration and is ineffective in inducing any oxidative stress. As this effect is strictly correlated with the mitochondrial permeability transition induction and the triggering of the pro-apoptotic pathway, AGM may be considered as a regulator of this type of cell death. Furthermore, polyamine transport is positively correlated with the rate of cellular proliferation. By increasing the expression of antizyme, a protein that inhibits polyamine biosynthesis and transport, AGM also exhibits a regulatory effect on cell proliferation. Methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosyl-l-methionine decarboxylase, displaying anticancer activity, is a structural analogue of the natural polyamine spermidine. MGBG has been extensively studied, preclinically as well as clinically, and its anticancer activity has been attributed to the inhibition of polyamine biosynthesis and also to its effect on mitochondrial function. Numerous findings have suggested that MGBG might be used as a chemotherapeutic agent against cancer.
Keywords: Biogenic polyamines; Quantum mechanical calculations; Agmatine; MGBG; Structure–activity relationships; Mitochondria
Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine by Autar K. Mattoo; Subhash C. Minocha; Rakesh Minocha; Avtar K. Handa (pp. 405-413).
Distribution of biogenic amines—the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)—differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (α and β), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd–Spm are positive regulators of cellular amino acid metabolism.
Keywords: Amino acids; Biogenic amines; Metabolome; Poplar cell cultures; Tomato
The polyamine transport system as a target for anticancer drug development by Andrew J. Palmer; Heather M. Wallace (pp. 415-422).
The vast majority of anticancer drugs in clinical use are limited by systemic host toxicity due to their non-specific side effects. These shortcomings have led to the development of tumour specific drugs which target a single-deregulated pathway or over expressed receptor in cancer cells. Whilst this approach has achieved clinical success, we have also learnt that targeting a single entity in cancer is rarely curative due to the large number of deregulated pathways, receptors and kinases which are also present, in addition to the target. An attractive alternative to improve targeting would be to harness the already established activity of known anticancer drugs by attaching them to a molecule that is transported into cancer cells via a selective transport system. One possibility for this approach is the polyamine pathway. This review provides a brief overview of the polyamine pathway and how, over the years, it has proved an exciting target for the development of novel anticancer agents. However, the focus of this article will be on the properties of the polyamine transport system and how these features could potentially be exploited to develop a novel and selective anticancer drug delivery system.
Keywords: Polyamine transport system; Targeted drug delivery; Anticancer therapy
Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria by V. Battaglia; S. Grancara; M. Mancon; C. Cravanzola; S. Colombatto; M. A. Grillo; G. Tempera; E. Agostinelli; A. Toninello (pp. 423-430).
The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force–flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux–voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I2 and the sharing with the polyamine transporter.
Keywords: Rat brain mitochondria; Agmatine; Kinetics; Polyamine; Transport; Imidazoline receptor
Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria by V. Battaglia; S. Grancara; J. Satriano; S. Saccoccio; E. Agostinelli; A. Toninello (pp. 431-437).
The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca2+-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (ΔΨ) drop. MPT is due to oxidative stress induced by the interaction of Ca2+ with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.
Keywords: Rat brain mitochondria; Agmatine; Mitochondrial permeability transition; Ca2+ ; Reactive oxygen species
Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos by Koichiro Shiokawa; Mai Aso; Takeshi Kondo; Jun-Ichi Takai; Junki Yoshida; Takamichi Mishina; Kota Fuchimukai; Tsukasa Ogasawara; Taro Kariya; Kosuke Tashiro; Kazuei Igarashi (pp. 439-449).
We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.
Keywords: S-adenosylmethionine decarboxylase (SAMDC); Maternal program of apoptosis; Polyamines; Ornithine; Amino acids; Amines; Ammonium ion; rRNA synthesis; Xenopus embryos
Structural biology of S-adenosylmethionine decarboxylase by Shridhar Bale; Steven E. Ealick (pp. 451-460).
S-adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and a subject of many structural and biochemical investigations for anti-cancer and anti-parasitic therapy. The enzyme undergoes an internal serinolysis reaction as a post-translational modification to generate the active site pyruvoyl group for the decarboxylation process. The crystal structures of AdoMetDC from Homo sapiens, Solanum tuberosum, Thermotoga maritima, and Aquifex aeolicus have been determined. Numerous crystal structures of human AdoMetDC and mutants have provided insights into the mechanism of autoprocessing, putrescine activation, substrate specificity, and inhibitor design to the enzyme. The comparison of the human and potato enzyme with the T. maritima and A. aeolicus enzymes supports the hypothesis that the eukaryotic enzymes evolved by gene duplication and fusion. The residues implicated in processing and activity are structurally conserved in all forms of the enzyme, suggesting a divergent evolution of AdoMetDC.
Keywords: Polyamines; Protein evolution; Putrescine activation; Cation–π interactions; Pyruvoyl cofactor
Down-regulation of hypusine biosynthesis in plasmodium by inhibition of S-adenosyl-methionine-decarboxylase by Robert Blavid; Peter Kusch; Joachim Hauber; Ute Eschweiler; Salem Ramadan Sarite; Sabine Specht; Susanne Deininger; Achim Hoerauf; Annette Kaiser (pp. 461-469).
An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Km i of 3 μM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 μM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 μM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials.
Keywords: AdoMETDC; EIF-5A; Malaria; SAM486A
Assessment of deoxyhypusine hydroxylase as a putative, novel drug target by B. Kerscher; E. Nzukou; A. Kaiser (pp. 471-477).
Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.
Keywords: Deoxyhypusine hydroxylase; Plasmodium ; Malaria; Drug target
Evidence for conformational changes in the yeast deoxyhypusine hydroxylase Lia1 upon iron displacement from its active site by Veridiana S. P. Cano; Francisco Javier Medrano; Myung Hee Park; Sandro R. Valentini (pp. 479-490).
The unique amino acid hypusine is formed exclusively in eIF5A by the successive action of deoxyhypusine synthase and deoxyhypusine hydroxylase (yeast Lia1, human DOHH). Although the first enzyme has been extensively studied, both Lia1 structure and the mechanism of action remain unclear. Hence, a multi-approach was used to evaluate Lia1 catalysis, metal/substrate binding, structural conformation and stability. Mutational analyses of Lia1 revealed fine differences in the mode of substrate binding between the human and yeast counterparts. Like human DOHH, recombinant Lia1 is an iron metalloenzyme. Iron is essential for enzyme activity since its loss renders the enzyme totally inactive. The separation of iron-free and iron-bound forms by gel filtration and native electrophoresis suggests differences in Lia1 tertiary structure related to the iron binding. The ability of Lia1 to undergo conformational changes prompted us to use a set of complementary spectroscopic approaches and SAXS to obtain detailed information on the processes underlying dissociation of iron from Lia1 at different levels of the protein organization. The additive effect of weak interactions, especially within the metal center, resulted in an active enzyme in a stabilized and compact three-dimensional fold. Loss of tertiary contacts upon iron displacement led to an elongated conformation of Lia1, in which the N- and C-terminal domains are no longer in close proximity to guarantee the proper orientation of the active groups within the active site pocket. Our results demonstrate an essential structural role for iron binding in addition to its contribution to the catalysis of hypusine formation in the eIF-5A precursor.
Keywords: eIF5A; Lia1; Hypusine; Deoxyhypusine hydroxylase; HEAT-repeat containing protein; Iron metalloenzyme; Structural analysis
Functional significance of eIF5A and its hypusine modification in eukaryotes by M. H. Park; K. Nishimura; C. F. Zanelli; S. R. Valentini (pp. 491-500).
The unusual basic amino acid, hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.
Keywords: Hypusine; eIF5A; Posttranslational modification; Polyamine; Deoxyhypusine synthase; Deoxyhypusine hydroxylase; Gene inactivation
Novel isosteric charge-deficient spermine analogue—1,12-diamino-3,6,9-triazadodecane: synthesis, pK a measurement and biological activity by Janne Weisell; Mervi T. Hyvönen; Jouko Vepsäläinen; Leena Alhonen; Tuomo A. Keinänen; Alex R. Khomutov; Pasi Soininen (pp. 501-507).
Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2–11.0 using two-dimensional 1H-15N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic pK a values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes.
Keywords: Polyamines; Spermine analogues; pK a ; NMR spectroscopy; Cell growth
Novel convenient synthesis of biologically active esters of hydroxylamine by Maxim A. Khomutov; Swati Mandal; Janne Weisell; Neiha Saxena; Alina R. Simonian; Jouko Vepsalainen; Rentala Madhubala; Sergey N. Kochetkov (pp. 509-517).
Alkylation of ethyl N-hydroxyacetimidate with readily available methanesulfonates of functionally substituted alcohols and subsequent deprotection of aminooxy group is a novel and convenient method to prepare functionally substituted esters of hydroxylamine with high overall yield. This approach is a good alternative to well-known reaction of N-hydroxyphthalimide with alcohols under the Mitsunobu conditions. The properties of ethoxyethylidene protection of aminooxy group on the contrary to that of N-alkoxyphthalimide group allow to perform a wide spectra of the transformations in the radical of N-protected hydroxylamine derivatives. This is essential for synthetic strategies consisting in the introduction of N-protected aminooxy group at one of the first steps of synthesis and subsequent transformations of the radical.The inhibitory effect of one of the newly synthesized compound, 1-guanidinooxy-3-aminopropane (GAPA), was compared with that of well-known inhibitors of ornithine decarboxylase namely, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA) on Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. GAPA, on the contrary with APA and DFMO, in micromolar concentrations, inhibited the growth of both amastigotes and promastigotes of sodium antimony gluconate-resistant forms of L. donovani.
Keywords: Hydroxylamines; Synthesis; Enzyme inhibitors; Leishmaniasis; Polyamines
Targeting polyamines and biogenic amines by green tea epigallocatechin-3-gallate by Esther Melgarejo; José Luis Urdiales; Francisca Sánchez-Jiménez; Miguel Ángel Medina (pp. 519-523).
Biogenic amines and polyamines are organic polycations derived from aromatic or cationic amino acids. They exert pleiotropic effects, more related to intercellular communication in the case of biogenic amines, and to intracellular signaling in the case of polyamines. The bioactive compound epigallocatechin-3-gallate (EGCG), a major component of green tea, has been shown to target key enzyme of biogenic amine and polyamine metabolic pathways. Herein, we review the specific effects of EGCG on concrete molecular targets of both biogenic amine and polyamine metabolic pathways, and discuss the relevance of these data to support the potential therapeutic interest of this compound.
Keywords: Polyamine; ODC; SSAT; DDC; HDC; Histamine; EGCG; Tea
Difluoromethylornithine inhibits hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone in cardiac cells by S. Cetrullo; A. Facchini; I. Stanic; B. Tantini; C. Pignatti; C. M. Caldarera; F. Flamigni (pp. 525-531).
Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (d,l)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.
Keywords: Aldosterone; Apoptosis; Cardiac cells; Difluoromethylornithine; Hypertrophy; Polyamines
Evaluation method for polyamine uptake by N 1-dansylspermine by Koichi Takao; Yoshiaki Sugita; Akira Shirahata (pp. 533-539).
Polyamine uptake by the polyamine transport system (PTS) in HTC cells was studied without the use of radioisotope-labeled polyamines. N1-Dansylspermine (DNS343) was selected as a candidate probe to examine the PTS. DNS343 was incorporated into HTC cells, and its distribution in the cells was confirmed by fluorescence microscopy. The incorporation of DNS343 via PTS was confirmed by a competition study with bis(3-aminopropyl)amine, which is incorporated into cells via the PTS. In addition, the temperature dependency of DNS343 uptake and studies with inhibitors of ornithine decarboxylase and proteoglycan synthesis supported the use of DNS343 as a fluorescent probe for the PTS. The kinetics studies for HTC cells treated with or without an ornithine decarboxylase inhibitor indicated that DNS343 uptake was saturable and that the apparent Km values for the PTS were approximately 1.5 μM in both types of cells at 37°C. Thus, we developed an assay method for the PTS by high-performance liquid-chromatography with DNS343. The inhibitory effect of polyamine analogs and related compounds on DNS343 uptake was then examined and discussed.
Keywords: Polyamine; Polyamine transport system; HTC cells; N1-dansylspermine; HPLC assay; Polyamine analog
Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts by Marco Govoni; Francesca Bonavita; Lisa M. Shantz; Carlo Guarnieri; Emanuele Giordano (pp. 541-547).
Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.
Keywords: H9c2 cells; Muscle differentiation; Ornithine decarboxylase; Polyamines; α-Difluoromethylornithine
Activated polyamine catabolism leads to low cholesterol levels by enhancing bile acid synthesis by Eija Pirinen; Helena Gylling; Paula Itkonen; Nagendra Yaluri; Sami Heikkinen; Marko Pietilä; Teemu Kuulasmaa; Maija Tusa; Marc Cerrada-Gimenez; Jussi Pihlajamäki; Leena Alhonen; Juhani Jänne; Tatu A. Miettinen; Markku Laakso (pp. 549-560).
Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase (SSAT) have significantly reduced plasma total cholesterol levels. In our study, we show that low cholesterol levels were attributable to enhanced bile acid synthesis in combination with reduced cholesterol absorption. Hepatic cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme catalyzing the conversion of cholesterol to bile acids, plays an important role in the removal of excess cholesterol from the body. We suggest that by reducing activity of Akt activated polyamine catabolism increased the stability and activity of peroxisome proliferator-activated receptor γ co-activator 1α, the critical activator of CYP7A1. This is supported by our finding that the treatment with SSAT activator, N 1,N 11-diethylnorspermine, reduced significantly the amount of phosphorylated (active) Akt in HepG2 cells. In summary, activated-polyamine catabolism is a novel mechanism to regulate bile acid synthesis. Therefore, polyamine catabolism could be a potential therapeutic target to control hepatic CYP7A1 expression.
Keywords: Spermidine/spermine N1-acetyltransferase; Cholesterol; Bile acids; Cholesterol 7α-hydroxylase; Peroxisome proliferator-activated receptor γ co-activator 1α; Akt
Exploring polyamine regulation by nascent histamine in a human-transfected cell model by H. Abrighach; I. Fajardo; F. Sánchez-Jiménez; J. L. Urdiales (pp. 561-573).
There are multiple lines of evidence suggesting interplay between histamine and polyamines in several mammalian cell types. However, the complex metabolic context makes it difficult to elucidate the mechanisms involved. Histamine’s effects can be elicited after its binding to any of the four subtypes of G-protein coupled histamine membrane receptors. In addition, intracellular histamine can also interfere with polyamine metabolism, since there are several metabolic connections between the synthesis and degradation pathways of both types of amines. In order to dissect the metabolic effects of intracellular histamine on polyamine metabolism, we chose a well-known cell culture line, i.e., the human embryonic kidney 293 cells (HEK-293 cells). Initially, we show that HEK-293 cells lack a polyamine metabolic response to extracellular histamine, even over a wide range of histamine concentrations. HEK-293 cells were transfected with active and inactive versions of human histidine decarboxylase, and changes in many of the overlapping metabolic factors and limiting steps were tested. Overall, the results indicate a regulatory effect of histamine on the post-transcriptional expression of ornithine decarboxylase and suggest that this effect is primarily responsible for the decrease in polyamine synthesis and partial blockade of cell-cycle progression, which should affect cell proliferation rate.
Keywords: Polyamines; Histamine; Histidine decarboxylase; Ornithine decarboxylase; Cell cycle
α-Methylspermidine protects against carbon tetrachloride-induced hepatic and pancreatic damage by Mervi T. Hyvönen; Riitta Sinervirta; Nikolay Grigorenko; Alex R. Khomutov; Jouko Vepsäläinen; Tuomo A. Keinänen; Leena Alhonen (pp. 575-581).
The role of polyamines in carbon tetrachloride (CCl4)-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl4 resulted in the induction of hepatic spermidine/spermine N 1-acetyltransferase (SSAT), accumulation of putrescine, reduction in spermine level and appearance of moderate hepatic injury within 24 h. Upon treatment with CCl4, transgenic rats overexpressing SSAT displayed induction of both hepatic and pancreatic SSAT, with subsequent accumulation of putrescine and decrease of both spermidine and spermine pools. Administration of CCl4 in SSAT transgenic rats induced not only massive hepatic injury, but also severe acute necrotizing pancreatitis. Pretreatment of the animals with catabolically stable functional polyamine mimetic, α-methylspermidine (MeSpd) prevented pancreatic and hepatic injury in SSAT rats and markedly reduced liver damage in syngenic animals. As assessed by immunostaining of proliferating cell nuclear antigen, MeSpd increased the amount of regenerating hepatocytes in both genotypes. These results show that CCl4 induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl4-induced hepatic and pancreatic damage and promotes tissue regeneration.
Keywords: Carbon tetrachloride; Spermidine/spermine N 1-acetyltransferase; Pancreatitis; Hepatotoxicity; Regeneration; Polyamines; Polyamine analogues; Liver cirrhosis
Cutaneous application of α-methylspermidine activates the growth of resting hair follicles in mice by Tekele M. Fashe; Tuomo A. Keinänen; Nikolay A. Grigorenko; Alex R. Khomutov; Juhani Jänne; Leena Alhonen; Marko Pietilä (pp. 583-590).
Recent studies using transgenic animals have revealed a crucial role for polyamines in the development and the growth of skin and hair follicles. In mammals, the growth of hair is characterized by three main cyclic phases of transformation, including a rapid growth phase (anagen), an apoptosis-driven regression phase (catagen) and a relatively quiescent resting phase (telogen). The polyamine pool during the anagen phase is higher than in telogen and catagen phases. In this study, we used α-methylspermidine, a metabolically stable polyamine analog, to artificially elevate the polyamine pool during telogen. This manipulation was sufficient to induce hair growth in telogen phase mice after 2 weeks of daily topical application. The application site was characterized by typical features of anagen, such as pigmentation, growing hair follicles, proliferation of follicular keratinocytes and upregulation of β-catenin. The analog penetrated the protective epidermal layer of the skin and could be detected in dermis. The natural polyamines were partially replaced by the analog in the application site. However, the combined pool of natural spermidine and α-methylspermidine exceeded the physiological spermidine pool in telogen phase skin. These results highlight the role of polyamines in hair cycle regulation and show that it is possible to control the process of hair growth using physiologically stable polyamine analogs.
Keywords: Alopecia; Polyamine; Skin; Spermidine
Developmental alterations in expression and subcellular localization of antizyme and antizyme inhibitor and their functional importance in the murine mammary gland by Y. Murakami; J. Suzuki; K. Samejima; T. Oka (pp. 591-601).
Ornithine decarboxylase (ODC), antizyme (AZ), and antizyme inhibitor (AIn) play a key role in regulation of intracellular polyamine levels by forming a regulatory circuit through their interactions. To gain insight into their functional importance in cell growth and differentiation, we systematically examined the changes of their expression, cellular polyamine contents, expression of genes related to polyamine metabolism, and β-casein gene expression during murine mammary gland development. The activity of ODC and AZ1 as well as putrescine level were low in the virgin and involuting stages, but they increased markedly during late pregnancy and early lactation when mammary cells proliferate extensively and begin to augment their differentiated function. The level of spermidine and expression of genes encoding spermidine synthase and AIn increased in a closely parallel manner with that of casein gene expression during pregnancy and lactation. On the other hand, the level of spermidine/spermine N 1-acetyltransferase (SSAT) mRNA and AZ2 mRNA decreased during those periods. Immunohistochemical analysis showed the translocation of ODC and AIn between the nucleus and cytoplasm and the continuous presence of AZ in the nucleus during gland development. Reduction of AIn by RNA interference inhibited expression of β-casein gene stimulated by lactogenic hormones in HC11 cells. In contrast, reduction of AZ by AZsiRNA resulted in the small increase of β-casein gene expression. These results suggested that AIn plays an important role in the mammary gland development by changing its expression, subcellular localization, and functional interplay with AZ.
Keywords: Mammary gland development; Polyamines; Ornithine decarboxylase; Antizyme; Antizyme inhibitor; β-Casein
Antizyme inhibitor 2: molecular, cellular and physiological aspects by Andrés J. López-Contreras; Bruno Ramos-Molina; Asunción Cremades; Rafael Peñafiel (pp. 603-611).
Polyamines are small organic polycations essential for cell proliferation and survival. Antizymes (AZs) are small proteins regulated by polyamines that inhibit polyamine biosynthesis and uptake in mammalian cells. In addition, antizyme functions are also regulated by antizyme inhibitors, homologue proteins of ornithine decarboxylase lacking enzymatic activity. There are two antizyme inhibitors (AZIN), known as AZIN1 and AZIN2, that bind to AZs and negate their effects on polyamine metabolism. Here, we review different molecular and cellular properties of the novel AZIN2 with particular emphasis on the role that this protein may have in brain and testis physiology. Whereas AZIN1 is ubiquitously found in mammalian tissues, AZIN2 expression appears to be restricted to brain and testis. In transfected cells, AZIN2 is mainly located in the endoplasmic reticulum–Golgi intermediate compartment and in the cis-Golgi network. AZIN2 is a labile protein that is degraded by the proteasome by a ubiquitin-dependent mechanism. Regarding its physiological role, spatial and temporal analyses of AZIN2 expression in the mouse testis suggest that this protein may have a role in spermiogenesis.
Keywords: Antizymes; Antizyme inhibitor 2; Polyamines; Ornithine decarboxylase like; Arginine decarboxylase; Spermiogenesis
Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism by Marc Cerrada-Gimenez; Jukka Häyrinen; Sisko Juutinen; Tuula Reponen; Juhani Jänne; Leena Alhonen (pp. 613-622).
We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N 1-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor γ co-activator 1α in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.
Keywords: Polyamines; Proteomics; SSAT; Liver; Transgenic mouse
Differential effects of cold, osmotic stress and abscisic acid on polyamine accumulation in wheat by Zita Kovács; Livia Simon-Sarkadi; Attila Szűcs; Gábor Kocsy (pp. 623-631).
The effects of cold, osmotic stress and abscisic acid (ABA) on polyamine accumulation were compared in the moderately freezing-sensitive wheat (Triticum aestivum L.) variety Chinese Spring (CS) and in two derived chromosome 5A substitution lines, CS(T. spelta 5A) and CS(Cheyenne 5A), exhibiting lower and higher levels of freezing tolerance, respectively. When compared with the other treatments, putrescine (Put) and spermidine (Spd) levels were much greater after cold treatment, spermine (Spm) following polyethylene glycol-induced (PEG) osmotic stress and Spm and cadaverine (Cad) after ABA treatment. During 3-week cold stress, the Put concentration, first exhibited a transient increase and decrease, and then gradually increased. These alterations may be due to changes in the expression of genes encoding the enzymes of Put synthesis. The Put content was higher in the freezing-tolerant chromosome 5A substitution line than in the sensitive one after 3 weeks of cold. In contrast to cold, ABA and PEG induced a continuous decrease in the Spd level when applied for a period of 3 weeks. The Spm content, which increased after PEG and ABA addition, was twice as high as that of Put during ABA treatment at most sampling points, but this difference was lower in the case of PEG. The Cad level, induced to a great extent by ABA, was much lower when compared with that of the other polyamines. The present experiments indicate that cold, osmotic stress and ABA have different effects on polyamines, and that some of these changes are affected by chromosome 5A and correlate with the level of stress tolerance.
Keywords: Abscisic acid; Cold stress; Osmotic stress; Polyamine; Wheat
Functional consequences of perturbing polyamine metabolism in the malaria parasite, Plasmodium falciparum by K. Clark; J. Niemand; S. Reeksting; S. Smit; A. C. van Brummelen; M. Williams; A. I. Louw; L. Birkholtz (pp. 633-644).
Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, α-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.
Keywords: Polyamines; Malaria; Plasmodium ; Parasites; Functional genomics; Difluoromethylornithine
Polyamine metabolism in Trypanosoma cruzi: studies on the expression and regulation of heterologous genes involved in polyamine biosynthesis by I. D. Algranati (pp. 645-651).
Biochemical studies have shown that Trypanosoma cruzi and Toxoplasma gondii are the only eukaryotic organisms so far described which are auxotrophic for polyamines. Both parasites are unable to carry out the de novo biosynthesis of putrescine, and therefore they need the addition of exogenous polyamines to the culture medium for their normal proliferation. Further investigations at the molecular level have demonstrated that the wild-type T. cruzi genome does not contain ornithine or arginine decarboxylase-like nucleic acid sequences, and that the corresponding genes have been presumably lost during evolution. Since T. cruzi behaves as a deletion mutant for ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes, this parasite has been selected to study the regulation of the expression of heterologous genes involved in polyamine biosynthesis in other organisms. The resulting transgenic parasites have been useful tools to analyze the different stages of gene expression after transformation, as well as the mechanisms of drug resistance induction and the post-translational processing of enzyme precursors.
Keywords: Polyamine auxotrophy; Trypanosoma cruzi ; Transgenic parasites; Gene expression; DFMO resistance; Post-translational processing
Critical role of transglutaminase and other stress proteins during neurodegenerative processes by Daniela Caccamo; Monica Currò; Salvatore Condello; Nadia Ferlazzo; Riccardo Ientile (pp. 653-658).
Proteolytic stress, resulting from the intracellular accumulation of misfolded or aggregated proteins, which exceed the capacity of the ubiquitin–proteasome system to degrade them, plays a relevant role in neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea. Most of toxic protein aggregates are characterised by the presence of isopeptide bonds (cross-links) catalysed by transglutaminase activity; further, several disease-specific proteins—tau, amyloid-beta, alpha-synuclein, huntingtin—are in vitro and/or in vivo substrates of transglutaminase 2. These findings suggest an important role for transglutaminase 2-mediated cross-linking reactions in neurodegeneration. Therefore, the use of transglutaminase activity inhibitors could ameliorate neuronal cell death. New therapeutic perspectives also arise from the possibility to prevent or reduce protein aggregation by enhancing the activation of heat shock proteins, which have been shown to be potent suppressors of neurodegeneration in cell cultures/animal models. Interestingly, some heat shock proteins have been shown to be in vitro or in vivo cross-linked by transglutaminase 2. These observations seem to suggest that transglutaminase activity could be involved in the stabilization of intracellular protein aggregates by interfering with proteasomal degradation of misfolded proteins. Further studies are needed to validate leading hypotheses and to open new prospects for developing therapeutic tools.
Keywords: Transglutaminases; Tissue transglutaminase; Heat shock proteins; Protein aggregates; Neurodegenerative diseases
Compatible and self-incompatible pollination in Pyrus communis displays different polyamine levels and transglutaminase activity by S. Del Duca; G. Cai; A. Di Sandro; D. Serafini-Fracassini (pp. 659-667).
The polyamine (PA) content and the transglutaminase (TGase) activity have been investigated in Pyrus communis pollination with compatible and self-incompatible (SI) pollen in order to deepen their possible involvement in the progamic phase of plant reproduction. The PA distribution as free, perchloric acid (PCA)-soluble and PCA-insoluble fractions in ungerminated (UGP), germinating pollen (GP), styles and pollinated styles with compatible and SI pollens is discussed in the light of a possible role during pollination. Generally, the conjugated PAs both in PCA-soluble and PCA-insoluble fractions were higher than the free form. Within the conjugated PAs, the PCA-insoluble ones were the highest with the exception of the not pollinated styles. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme, never checked before in styles and pollinated styles, was examined. In the SI styles, the TGase activity is higher in comparison to style-pollinated with compatible pollen, and high molecular mass cross-linked products were formed, suggesting an involvement of TGase in SI response. This is the first evidence on the presence of this enzyme activity in not pollinated and pollinated styles.
Keywords: Polyamines; Transglutaminase; Pollen; Reproduction; Self-incompatibility; Rosaceae
Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films by Prospero Di Pierro; Loredana Mariniello; Angela Sorrentino; Reynaldo Villalonga; Belkis Chico; Raffaele Porta (pp. 669-675).
Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.
Keywords: Putrescine; Transglutaminase; Edible films; Pectin; Alginate; Crosslink