Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Activation of a novel isoform of methionine adenosyl transferase 2A and increased S-adenosylmethionine turnover in lung epithelial cells exposed to hyperoxia

S-Adenosylmethionine (SAM, AdoMet) is the most important methyl donor used for synthesis of nucleic acids, phospholipids, creatine, and polyamines and for methylation of many bioactive molecules. The metabolic response of the lung to oxidative stress of hyperoxia requires increased RNA and protein synthesis for energy metabolism, growth arrest, and antioxidant defense. We studied the production of SAM and other aspects of methionine metabolism in lung epithelial cells exposed to hyperoxia. Human lung epithelial-like (A549) and primary small airway epithelial (SAE) cells were exposed to normoxia (21% O(2)) or hyperoxia (95% O(2)). Cell methionine and S-adenosylmethionine content increased in response to hyperoxia in SAE and A549 cells. Because methionine adenosyl transferase (MAT) is the rate-limiting enzyme of the pathway, we examined the expression of a lung epithelial isoform of MAT 2A in hyperoxia. Western blots revealed a novel MAT 2A isoform expressed in both cell types, with a lower molecular mass than that described in Jurkat cells. Cloning and sequencing of the MAT 2A cDNA revealed one silent nucleotide substitution compared to that expressed in Jurkat. The lower mass of MAT 2A in both lung epithelial cells indicated that the absence of the major posttranslational modification of MAT 2A found in Jurkat. MAT 2A protein progressively increased during hyperoxic exposure in both transformed and primary lung epithelium. Increased flux of (13)C-labeled methionine to S-adenosylhomocysteine (SAH) in A549 demonstrated that SAM's methyl group was utilized, and increased formation of cystathionine indicated that at least part of SAM generated was directed toward cysteine/GSH in the transsulfuration pathway. These results indicate activation of MAT 2A and the transmethylation pathway in the metabolic response to hyperoxia in lung epithelium.     

Suppression of TNF-alpha production by S-adenosylmethionine in human mononuclear leukocytes is not mediated by polyamines

Endotoxin-induced cytokine production is an important mechanism in the development of several types of liver damage. Methionine, some of its precursors and metabolites were reported to have protective effects against such injury. The aim of this study was to investigate whether methionine, its precursors or metabolites [phosphatidylcholine, choline, betaine, S-adenosylmethionine (SAM)] have a modulating effect on tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated human mononuclear leukocytes and whether SAM-dependent polyamines (spermidine, spermine) are mediators of SAM-induced inhibition of TNF-alpha synthesis. Methionine and betaine had a moderate stimulatory effect on TNF-alpha production, whereas phosphatidylcholine (ID(50) 5.4 mM), SAM (ID(50) 131 microM), spermidine (ID(50) 4.5 microM) and spermine (ID(50) 3.9 microM) had a predominantly inhibitory effect. Putrescine did not alter TNF-alpha release. Inhibitors of polyamine synthesis that blocked either putrescine (difluoromethylornithine) or spermine (CGP48664A) production did not affect TNF-alpha synthesis. Endotoxin stimulation of leukocytes did not alter the intracellular levels of polyamines. In addition, supplementation with SAM did not change the intracellular concentration of either polyamine measured. We conclude that phosphatidylcholine-induced immunosuppression is not caused by methionine and polyamines are not involved in SAM-induced inhibition of TNF-alpha production. The limitation of TNF-alpha release by spermidine is specific and is not due to its conversion into spermine.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of α-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.     

Micro determination of polyamines with snake venom oxidase.

An accurate micromethod suitable for the assay of polyamines in concentrations of 2 to 30 nmol is described. It is based on the oxidation of polyamines by purified fractions of crude l-amino acid oxidase from Russell's viper venom. By a combination of two enzyme fractions, one which oxidizes polyamines and amino acids (AAP) and another which oxidizes only amino acids (AA), the technique can accurately determine polyamine concentrations in extracts of sera which may not be free of amino acids. Experiments described show 90 to 100% recovery of added polyamines in the presence of varying amounts of amino acids. Polyamines added to serum also showed recoveries ranging from 96 to 98%. The enzymes do not oxidize histamine and epinephrine and are very stable. The method does not require sophisticated equipment and is suitable for screening of large number of clinical samples to assess the importance of polyamines as a diagnostic test or their prognostic value in diseases like cancer.     

Biosynthesis of 7,8-diaminopelargonic acid, a biotin intermediate, from 7-keto-8-aminopelargonic acid and S-adenosyl-L-methionine

Resting cells of Escherichia coli strain D302(bioD302) can synthesize 7,8-diaminopelargonic acid from 7-keto-8-aminopelargonic acid. The product of this aminotransferase reaction has been identified by paper chromatography and electrophoresis. Glucose enhances the vitamer yield twofold. Of the 19 amino acids tested as amino donors, only methionine proved to be significantly stimulatory. In cell-free extracts, however, methionine was completely inactive unless both adenosine triphosphate (ATP) and Mg(2+) were present. S-Adenosyl-l-methionine (SAM) was about 10 times more effective than methionine, ATP, and Mg(2+). The optimal conditions for the reaction were determined, and substrate inhibition was found for 7-keto-8-aminopelargonic acid. It has been possible to eliminate certain impurities as amino donors in the commercial preparation of SAM and those that may arise in enzymatic reactions in which SAM is a substrate. The direct participation of SAM in the aminotransferase reaction seems a likely possibility.     

Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.     

Functions of polyamine acetylation

Acetylation is a means to decrease the net positive charge of the polyamines and thus liberate polyamines from anionic binding sites. The acetyl derivatives can be removed from the cells by transport and catabolism. Intracellular polyamine metabolism can be formulated as a cyclic process, which explains the transformation of one polyamine into another. As a net result, this pathway metabolizes (in an energy-requiring manner) methionine to 5'-deoxy-5'-methylthioadenosine and beta-alanine, and thus appears to be futile. It is suggested that the cyclic process is necessary for the precise control of cellular polyamine concentrations, as it allows relatively rapid spermine and spermidine concentration changes, in spite of a slow basal turnover rate. For the regulation of cellular polyamine metabolism, two decarboxylases, L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase; the cytosolic acetyl-CoA:spermidine/spermine N1-acetyltransferase; and a polyamine transport system are required. The activity of the nuclear acetyltransferase is assumed to be the rate-limiting enzyme of nuclear polyamine turnover. The complexity and high level of sophistication of polyamine regulation is strong evidence for the important functional significance of the natural polyamines.     

PHYSIOLOGICAL DEPENDENCE OF BRAIN METHIONINE AND S-ADENOSYLMETHIONINE CONCENTRATIONS ON SERUM AMINO ACID PATTERN

Abstract— Animals maintained on rat chow and water ad libitum in quarters illuminated for 12 h/day show diurnal rhythms in serum methionine and brain S -adenosylmethionine (SAM) concentrations. Brain methionine exhibits no such variation, nor does the ratio of serum methionine to the serum concentrations of six neutral amino acids which are believed to compete with methionine for uptake into brain. Administration of methionine to rats in doses that elevate serum methionine, but keep it within the daily physiological range, significantly increases brain concentrations of both methionine and SAM. The acute feeding of either a protein-free or a 40% casein meal also increases brain methionine and SAM, but does not affect serum methionine; however, both diets also increase the ratio of serum methionine to tyrosine, an amino acid whose postprandial concentration is indicative of the concentrations of the other amino acids that compete with methionine for transport into brain. These findings suggest that brain methionine levels increase physiologically after eating as a result of changes in the serum amino acid pattern. Furthermore, such naturally occurring increases in brain methionine appear to be associated with elevations in brain SAM.     

Functional analysis of <Emphasis Type="Italic">OsPUT1</Emphasis>, a rice polyamine uptake transporter

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.     

Methionine is required for cAMP‐PKA‐mediated morphogenesis and virulence of Candida albicans

Candida albicans is a major human fungal pathogen, causing superficial, as well as life‐threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast‐to‐hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine‐induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S‐adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino‐propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine‐induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP‐PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.     

Polyamine flux analysis by determination of heavy isotope incorporation from 13C, 15N-enriched amino acids into polyamines by LC-MS/MS

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.     

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.     

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10–20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5–20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17–44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.Abbreviations MET methionine - SAM S-adenosylmethionine - SAH S-adenosylhomocysteine - SZ streptozotocin - BBW BB Wistar - LNAA large neutral amino acids - BCAA branchedchain amino acids - MET:BCAA methionine to branched-chain amino acid ratio - MET:LNAA methionine to large neutral amino acid ratio     

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

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

Polyamine synthesis from proline in the developing porcine placenta

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.     

Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.