No influence of the embryonic axis on the development of diamine oxidase in pea cotyledons

A reexamination has been made for the supposed regulation of pea (Pisum sativum cv Alaska) cotyledonary diamine oxidase (EC 1.4.3.6) activity by the embryonic axis. When dry cotyledons from which the embryo and testa have been removed surgically are imbibed by soaking in water, there is little increase of the enzyme activity during subsequent incubation on filter paper. However, if the dry cotyledons are imbibed and maintained on filter paper from the first, the increase of the enzyme activity is similar to that in the intact seedling. Thus, rapid imbibition of the isolated dry cotyledons is responsible for repression of enzyme development, and a role for the axis need not be invoked.     

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.     

The oxidation of monodansyldiamines by pea seedling diamine oxidase

In the oxidation of a homologous series of monodansyldiamines by pea seedling diamine oxidase, monodansylcadaverine was the best substrate. Monodansyldiaminohexane was oxidized at 74% of the rate with monodansylcadaverine, and monodansylputrescine and monodansyldiaminopropane were oxidized only very slowly. The optimum pH for the oxidation of monodansylcadaverine was 8.5, and the K_m 2.4 × 10^?4 M. Under optimum conditions, putrescine was oxidized eleven times faster than monodansylcadaverine. Oxidation of monodansylcadaverine by diamine oxidase, and the exhaustive dansylation of lysine in equivalent amounts ultimately showed equal fluorescence in the dansyl-5-aminovaleraldehyde formed, indicating stoichiometric conversion to this product in both reactions.     

Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate     

Cryoenzymology and spectrophotometry of pea seedling diamine oxidase

Diamine oxidase follows bi-ter ping-pong kinetics, with an intermediate, "reduced" free-enzyme form being generated after the anaerobic conversion of amine to aldehyde. Visible spectra of diamine oxidase reacting at subzero temperatures provide evidence that this intermediate enzyme form is obtained via several other intermediates and that the environment of the Cu(II) changes dramatically during the course of the reaction [even though it is not reduced to Cu(I) during the catalytic cycle]. The spectrum of this form of diamine oxidase, which is obtained 0.5--2 h after the addition of amine at -5 to -15 degrees C, is independent of substrate, is identical with that obtained by anaerobic addition of substrate at room temperature, and provides evidence for a direct interaction of Cu(II) with the organic cofactor of the enzyme. This interaction is apparently charge transfer in nature. Upon removal of Cu(II) from the native enzyme, one obtains spectral evidence that the organic cofactor is still present. However, removal of the Cu(II) from the reduced (intermediate) enzyme form yields a featureless enzyme spectrum and a Cu(II)--chelate complex which contains a new ligand, which is presumably the second prosthetic group.     

Regulation of diamine oxidase activity in germinating pea seeds

Diamine oxidase present in the cotyledons of germinating pea seeds is induced by putrescine, spermidine and ornithine. Auxins inhibit enzyme synthesis in cotyledons only in the presence of embryo. Cycloheximide inhibits the synthesis of the cotyledon enzyme but has no effect on the embryo enzyme. 5-Fluorouracil inhibits the synthesis of both cotyledon and embryo enzymes.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Optimized preparation and determination of pea seedling diamine oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Effect of ethrel and chloroethanol on pea diamine oxidase activity

The activity of cotyledon and embryo diamine oxidase was reduced by feeding ethrel and chloroethanol to the seedlings. The inhibitory effect of 2,4-D on the activity of enzyme in the cotyledon which may be mediated through ethylene was reversed by exposure of seeds to red light.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

The formation of intercellular spaces in the cotyledons of developing and germinating pea seeds

Summary Electron microscopic observations have shown that the intercellular spaces in the storage parenchyma of the cotyledons of pea (Pisum sativum L.) seeds arise schizogenously. The wall segments adjoining future intercellular spaces display a triple zonation and contain regions with electron-dense material. Space formation starts at the central point of contact between several cells and spreads then further up to the electron-dense, intra-wall structures. As a result of this the electron-dense material is found again in the corners of intercellular spaces. It is proposed that the intra-wall structures may have an important function in limiting the schizogenous process. The localization of the intercellular spaces is thus predetermined. The amount of electron-dense material in their corners increases considerably during further development of the embryo. During germination wall segments between two intercellular spaces diverge resulting in a fusion of several spaces.     

1,4-Diamino-2-butyne as the mechanism-based pea diamine oxidase inhibitor.

1,4-Diamino-2-butyne is a mechanism-based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics Km = 1 mM like a substrate, but its interaction leads to time-dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4-diaminobutane and the competitive inhibitor 1,4-diamino-2-butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4-diamino-2-butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4-diamino-2-butyne is a mechanism-based inactivator with number of turnovers, r = 17 and characteristic constants K' = 0.32 mM and k(in) = 4.89 min-1.     

The effect of water stress on indoleacetic Acid oxidase in pea plants

Activity of indoleacetic acid oxidase was shown to increase following a period of water stress. Two fractions of indoleacetic acid oxidase were extracted from plant extracts. Similarly, two protein peaks (determined by ultraviolet absorption) were isolated. One peak, associated with an indoleacetic acid oxidase peak, increased following water stress. The second peak, not associated with extractable indoleacetic acid oxidase, decreased after water stress. The results are discussed in terms of general growth effects.     

A storage role for albumins in pea cotyledons

Abstract It is widely held that the albumins of pea seeds are mainly enzyme proteins. In the present study of Pisum sativum cv. Greenfeast, certain major polypeptides confined to the albumin fraction from the cotyledons were shown to be degraded in vivo following germination, thus functioning as a reserve of amino nitrogen and carbon skeletons. A general definition of seed storage proteins is proposed, to encompass any protein present in major quantity in the seed which is degraded following imbibition, and for which no other function may be demonstrated.     

Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase

The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of K(m) and catalytic constant (k(c)) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed.     

Carnitine content of pea seedling cotyledons

Using a radioactive assay for the determination of carnitine, the amount in pea cotyledons was shown to vary with age.     

Effect of the pea embryo on formation and degeneration of the mitochondrial membrane in cotyledons during germination

The effect of removal of the embryo on the properties of mitochondriain pea cotyledons was investigated. During imbibition of theseeds, mitochondrial activity was enhanced in the cotyledons.In later stages of germination, respiratory activity of themitochondria decreased gradually, and no response of the mitochondriato exogenous ADP was observed. Moreover, considerable activityof cytochrome oxidase wasrecovered in the post-mitochondrialfraction. Mitochondrial fractions isolated from senescent cotyledonscontained only fragmented particles of mitochondria. On theother hand, in cotyledons excised from the seeds and cultivatedunder wet condition, the initial development of mitochondriademonstrated in the attached cotyledons was suppressed. However,respiratory activity of the mitochondria increased in the laterstages of cultivation. The mitochondria remained unfragmentedand responded to exogenous ADP during all stages of cultivation.Also, a change in the density of mitochondria which occurredin the germinating attached cotyledons was delayed in the cultivatedexcised cotyledons. (Received February 27, 1973; )     

Competition of homologous substrates, putrescine and cadaverine, in the reaction catalyzed by pea diamine oxidase.

The determination of diamine oxidase activity with the ninhydrin reagent was used for monitoring of simultaneous oxidation of two homologous substrates, putrescine and cadaverine, which give different colour products (519 and 417 nm). We measured the reaction rates of oxidation of both substrates in different proportion and compared them with the total reaction rate determined by the guaiacol method. The substrates show competition with inhibition constants of putrescine against cadaverine of 0.14 mmol.l-1 and cadaverine against putrescine of 6.4 mumol.l-1.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.