Amine levels in Lathyrus sativus seedlings during development

In growing Lathyrus sativus seedlings, the levels of DNA, RNA and protein markedly decreased in the cotyledons and progressively increased in the embryo-axis. In cotyledons, spermidine and spermine contents were substantially reduced while those of agmatine and putrescine were sharply increased. By contrast the embryo-axis progressively accumulated relatively larger amounts of agmatine, homoagmatine. putrescine, cadaverine, spermidine and spermine in parallel with similar changes in its DNA, RNA and protein content. While the cotyledons contained ca 50% of the total agmatine and putrescine present in the plant embryo by day 10, the embryo-axis, though representing less than 20% of the dry wt, contained 90 and 75% of total cadaverine and homoagmatine respectively of the seedlings. Spermidine and spermine levels of this tissue were also comparatively higher, being of the order of 80 and 50% respectively of the total. The root and shoot portions of the embryo-axis also exhibited a similar relationship between changes in DNA, RNA and protein and all the above amines during development. However, the polyamine content of the shoots was relatively higher than those of the roots during the growth period.     

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when ¹⁴C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when ¹⁴C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.     

Diamine oxidase from Lens esculenta seedlings: Purification and properties

A diamine oxidase (DAO) (EC 1.4.3.6) has been purified to homogeneity from lentil seedlings. The purified protein has a MW of 154 000 and is composed of two apparently identical subunits. It contains two CU²⁺ atoms and one carbonyl-like group per mol. The purified enzyme is pink-red in concentrated solution and shows a broad, well-defined, absorption band in the visible region centered at 498 nm. The ESR spectrum is typical of Cu²⁺ in a tetragonal symmetry. The enzyme oxidizes only aliphatic diamines and spermidine with formation of the corresponding aldehydes, hydrogen peroxide and ammonia. Putrescine and cadaverine are oxidized most rapidly and the oxidation rate decreases when longer diamines are tested.     

Polyamine oxidation by enzymes from Hordeum vulgare and Pisum sativum seedlings

The properties of the amine oxidases of barley leaves and pea seedling cotyledons have been compared using a colorimetric assay in which the hydrogen p     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Homoagmatine from Lathyrus sativus seedlings

A new guanidino amine has been isolated from Lathyrus sativus seedlings and chararterized as homoagmatine on the basis of various physico-chemical criteria including IR spectrum and comparison with that chemically synthesized. Homoagmatine is accumulated in the embryos axis while its precursor, homoarginine, is lost from the cotyledons. However, there was a progressive increase in homoarginine content of the embryo axis during development. Since the amine content of the whole seedlings corresponded to nearly 20–25 % of net decrease in homoarginine levels, it is concluded that the catabolism of homoarginine through homoagmatine represents a major pathway of metabolism of the arnino acid.     

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.     

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l-lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn²⁺ or Fe²⁺ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The K_m value l-homoarginine was 3·33 mM and for l-lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.     

Immunoaffinity purification and characterization of diamine oxidase from Cicer

Antiserum specific for diamine oxidase (DAO;EC 1.4.3.6) from Lens culinaris cross-reacted with DAO from several other members of the Leguminosae when tested by agar double diffusion. Antibodies purified by affinity chromatography were used to make an immunoadsorbent for the one-step purification of DAO from various species of the Leguminosae. This technique has made it possible to purify in one step the already characterized DAO from pea and lentil, and the unknown diamine oxidase from Cicer arietinum. This enzyme was partially characterized; it showed a pH optimum of 7.5 with putrescine as substrate and followed typical Michaelis-Menten kinetics with a K_m of 2.4 × 10^?4 M. Copper ligands and carbonyl group-directed reagents inhibited the enzyme.     

The allantoinase of Lathyrus sativus

The presence of a stable allantoinase in Lathyrus sativas and its de novo synthesis at a maximal rate in the first 48 hr of germination have been demonstrated. The plumule and radicle together exhibited highest enzyme activity. L. sativas allantoinase has been purified nearly 35-fold. The purified enzyme was optically active around pH 7.5, did not require any metal ion for activity and exhibited a K_m of 2·56 mM for (±)-allantoin, and an activation energy of 5·6 kcal/mol. Unlike other plant allantoinases, the L. sativus enzyme is highly specific for (±)-allantoin and is shown to be a sulfhydryl enzyme which apparently exists in a stable form in vivo obviating the need for added sulfhydryl compounds for maximal activity.     

Inhibitor of diamine oxidase in cotyledons of groundnut seedlings

The apparent absence of diamine oxidase in extracts of cotyledon of germinating groundnut seeds is due to the presence of a natural inhibitor. The inhibitor was associated with the lipid in the upper layer obtained on centrifugation of the cotyledon extract. It was non-dialysable, thermolabile and was inactivated by TCA and Triton X-100. The inhibitor was detected in the cotyledon extracts from early stages of seed development and was present up to 20 days after germination.     

Replacement of Mg2+ by polyamines in the aminoacylation of tRNA from Phaseolus

The ability of putrescine, spermidine and spermine to replace Mg²⁺ ions in the charging reaction of tRNA was estimated for seventeen amino acids. The polyamines promoted only the transfer reaction in the case of Leu, Ile, Val, Tyr and Arg. A synergistic effect was observed when spermine was added to a suboptimal concentration of Mg²⁺ (charging at only 5% of the optimal level). This synergistic effect was not observed for Ala, Asp-NH₂, His, Lys and Ser. Kinetic studies showed a slower aminoacylation rate in those experiments when spermine and Mg⁺² (at 5% of the Mg²⁺ optimal concn) were used together than with Mg²⁺ (at the optimal concn) alone.     

Further properties of the polyamine oxidase of barley leaves

Polyamine analogues have been studied as potential inhibitors or substrates of barley leaf polyamine oxidase. NH₂(CH₂)₃NH(CH₂)₁₀NH₂ was particularly effective as an inhibitor of spermine oxidation at pH 4·5 (K_i = 5 × 10^?6 M). Methylglyoxal-bis(guanylhydrazone) inhibited spermine oxidation only slightly (K_i = 10^?4 M). Activity with the polyamine analogues as substrates was generally 10% or less of the activity with spermine. The K_m for oxygen was 3 × 10^?4 M. The K_m for spermine oxidation was independent of oxygen concentration. Using the N-methyl-2-benzothiazolone hydrazine reagent, 1-(3-aminopropyl)pyrroline was shown to be formed stoichiometrically by the enzyme on oxidation of spermine. The enzyme will not function as a dehydrogenase in the presence of oxygen with either potassium ferricyanide or dichlorophenolindophenol as electron acceptors. Activity in the leaves increased with age, up to 4 weeks. In the leaves of 11-week-old plants activity was lower than in leaves of 1-week-old plants. The enzyme was mainly associated with an easily-sedimented particulate fraction, and relatively small proportions were found in the cell wall or soluble fractions.     

Identification of cadaverine in Pisum sativum

Cadaverine has been identified in normal young peas and in saline grown (necrotic) peas by GLC and MS. Its concentration was at least of 5 gm/g fresh plant in both cases. Analysis of the alkaloidal extracts failed to reveal any lupinine, anabasine or other pyridine alkaloids in normal or necrotic, salt grown, peas.     

Changes in polyamine contents during root and nodule growth of Phaseolus mungo

In Phaseolus mungo seeds, polyamine content increased during early germination, being maximum after 24 hr; and the arginine decarboxylase showed a peak after 18 hr. During nodule initiation and growth two peaks of polyamine contents were noted-the first being 2 weeks after nodule initiation and a second one after 5 weeks. Arginine decarboxylase activity also followed the same pattern. In the roots the polyamine concentration as well as arginine decarboxylase increased up to week 2 after sowing followed by a gradual decrease. Estimation of RNA, DNA and protein contents showed a pattern similar to that of the polyamines.     

Polyamine oxidase from Zea mays shoots

Polyamine oxidase of maize shoots purified 10-fold had a pH optimum of 6·3 with spermidine as substrate, and K_m of 6 × 10^?4 M. The enzyme was inhibited by the acridine compounds quinacrine, 6,9-diamino-2-ethoxyacridine and acriflavin, but carbonyl reagents, typical thiol inhibitors and copper-binding agents were without effect. Inhibition by quinacrine was reversed by FMN and FAD. Furthermore, about 50 % of the activity of the apoenzyme was restored by the addition of FAD, but not by FMN or riboflavin, indicating that the maize polyamine oxidase is an FAD-dependent flavoprotein.     

Polyamines in barley seedlings

Polyamine levels in barley seedlings grown in the dark or in diurnal illumination have been determined, by direct dansylation, 3, 6 and 12 days after g     

Effect of polyamines on ribonuclease activity of rice (Oryza sativa L.)

The RNA content and RNase activity were determined during maturation and germination of rice seeds. The RNA content reached a maximum after the 16th day from anthesis but RNase activity steadily increased up to the last stage of maturation. During germination RNA content was greatest after 24 hr and associated with a very low level of RNase activity. Maximum RNase activity was observed at 72 hr from germination and it afterwards gradually declined. During germination, exogenous application of polyamines decreased the level of RNase activity. RNase was partially purified (448-fold) from 72 hr germinated embryonic axis. Effects of polyamines and other divalent cations were observed on the purified enzyme.     

An amine oxidase in seedlings of <Emphasis Type="Italic">Papaver somniferum</Emphasis> L.

Amine oxidase (AO) from 4-d-old seedlings of Papaver somniferum L. (Papaveraceae) was purified (58-fold) by using ammonium sulphate precipitation and chromatography on Sephadex G-150 and HA-Ultrogel columns. The most readily oxidized substrate was tyramine and other aromatic amines, while aliphatic amines cadaverine and putrescine were oxidized more slowly. Cu chelating and carbonyl reagents are the most effective inhibitors of poppy amine oxidase. Immunoblotting analysis showed cross reactivity of AO protein from poppy seedlings with polyclonal antisera against AO from pea. Obtained Mr value for AO from poppy (83 kDa) corresponds to that of copper AOs (75 – 90 kDa). These results suggest that the amine oxidase from poppy seedlings is a copper containing and tyramine specific AO.This work was supported by the grant of Slovak Grant Agency (VEGA 1/1197/04) and by the Comenius University, Faculty of Pharmacy Grant (FaF UK/1191/2002).