Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.     

Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

Functional characterization of allantoinase genes from Arabidopsis and a nonureide-type legume black locust

The availability of nitrogen is a limiting factor for plant growth in most soils. Allantoin and its degradation derivatives are a group of soil heterocyclic nitrogen compounds that play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in plants. Allantoinase is a key enzyme for biogenesis and degradation of these ureide compounds. Here, we describe the isolation of two functional allantoinase genes, AtALN (Arabidopsis allantoinase) and RpALN (Robinia pseudoacacia allantoinase), from Arabidopsis and black locust (Robinia pseudoacacia). The proteins encoded by those genes were predicted to have a signal peptide for the secretory pathway, which is consistent with earlier biochemical work that localized allantoinase activity to microbodies and endoplasmic reticulum (Hanks et al., 1981). Their functions were confirmed by genetic complementation of a yeast mutant (dal1) deficient in allantoin hydrolysis. The absence of nitrogen in the medium increased the expression of the genes. In Arabidopsis, the addition of allantoin to the medium as a sole source of nitrogen resulted in the up-regulation of the AtALN gene. The black locust gene (RpALN) was differentially regulated in cotyledons, axis, and hypocotyls during seed germination and seedling growth, but was not expressed in root tissues. In the trunk wood of a mature black locust tree, the RpALN gene was highly expressed in the bark/cambial region, but had no detectable expression in the sapwood or sapwood-heartwood transition zone. In addition, the gene expression in the bark/cambial region was up-regulated in spring and fall when compared with summer, suggesting its involvement in nitrogen mobilization.     

Immunoaffinity purification and comparison of allantoinases from soybean root nodules and cotyledons.

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Glycine max [L] Merr.). We characterized and compared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a Km for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. In addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70 degrees C, decreased gradually with heating to 85 degrees C, and was lost at 90 to 95 degrees C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified its purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Allantoinase from Mung Bean Seedlings

Mung bean allantoinase was purified sixty folds by calcium phosphate gel treatment, ammonium sulfate fractionation and acetone precipitation. The purified allantoinase hydro-lyzed allantoin to allantoic acid almost completely and the reaction had a broad pH optimum between 7.5 and 8.3. The accumulation of allantoic acid during the germination of mung bean was also noted. The allantoic acid content of seedlings was higher in hypocotyl than in leaf and root.     

Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO(2), and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 microm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum.     

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg⁻¹, which corresponds to a turnover number of 1,055 min⁻¹. The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K _m value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.     

Allantoinase and allantoicase synthesis in Pseudomonas aerguinosa.

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5.) and allanoicase (allantoate amidinohydrolase, EC 3.5.3.4) of Pseudomonas aeruginosa are inducible enzymes, whose syntheses are enhanced by the presence of allantoin, allantoate, ureidoglycolate, N-carbamoyl-L-asparagine, N-carbamoyl-L-aspartate, hydantoate, and diureidomethane. For each compound a specific ratio between the activities of allantoinase and allantoicase was obtained. The synthesis of these enzymes is not coordinately controlled. N-Carbamoyl-L-aspartate, hydantoate, and diureidomethane are gratuitous inducers.     

Metal ion dependence of recombinant Escherichia coli allantoinase

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess approximately 1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (k(cat) = 34.7 min(-1)) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (k(cat) values of 5,000 and 28,200 min(-1) and 200 min(-1), respectively). These rates and corresponding K(m) values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K(i) differences for the zinc and cobalt species (237 and 795 micro M, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.     

Evolution of allantoinase and allantoicase involved in urate degradation in liver peroxisomes. A rapid purification of amphibian allantoinase and allantoicase complex, its subunit locations of the two enzymes, and its comparison with fish allantoinase and allantoicase

Allantoinase and allantoicase are located in the same protein molecule in amphibian liver, whereas the two enzymes are different proteins in marine fish and invertebrate liver (Takada, Y., and Noguchi, T. (1983) J. Biol. Chem. 258, 4762-4764). The amphibian enzyme was rapidly purified from frog liver by using its following characteristics. 1) The enzyme binds to the intracellular membranes in the hypotonic solution. 2) The membrane-bound enzyme is not solubilized by the detergent. 3) The membrane-bound enzyme is solubilized by oxaloacetate. The electrophoresis of the purified enzyme gave a single protein band in the absence of sodium dodecyl sulfate, and gave two protein bands with molecular weights of 48,000 and 54,000, respectively, in the presence of sodium dodecyl sulfate. With a specific antibody raised against each subunit, allantoinase activity was found to be from the large subunit, and allantoicase activity to be from the small subunit. This amphibian allantoinase and allantoicase complex was compared with allantoinase and allantoicase purified from fish liver. Fish allantoinase was a single peptide and fish allantoicase was composed of two identical subunits. Fish allantoinase had an identical molecular weight with amphibian large (allantoinase) subunit and the subunit of fish allantoicase with amphibian small (allantoicase) subunit. These results suggest that the evolution of fish to amphibian resulted in the dissociation of allantoicase into subunits and in the association of allantoinase with allantoicase. The two enzymes are lost by further evolution.     

Allantoinase from nodules of pigeonpea (Cajanus cajan)

Allantoinase was purified about 10-fold from nitrogen fixing root nodules of pigeonpea (Cajanus cajan) using (NH₄)₂S0₄ fractionation and chromatography on Sephadex G-100. The purified preparation showed a specific activity of 1.73 nkat/mg protein, M_r of 125 000, pH optimum between 7.5 and 7.7 and K_m of 13.3 mM. The enzyme was heat stable up to 70dg and metal ions, except Hg²⁺, had no effect on the enzyme activity. The enzyme was inhibited significantly by reducing agents. Amino acids, ammonium, nitrate, potential precursors of allantoin and a number of other intermediate metabolites of ureide biosynthetic pathway had no effect on enzyme activity. It is suggested that allantoinase is unlikely to regulate the production of ureides in the nodule tissue.     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.

Labeling studies using ¹⁴CO₂ and ¹⁵N₂ and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.

Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.

Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.

     

Allantoinase from shoot tissues of soybeans

Allantoinase, catalysing the hydrolysis of allantoin to allantoic acid, was isolated from leaves and fruits of soybeans. The enzyme was only partially     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species. The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

Allantoinase activity and ureide content of mesophyll and paraveinal mesophyll of soybean leaves

Paraveinal mesophyll (PVM) is a specialized soybean (Glycine max Merr.) leaf tissue which represents a significant biochemical compartment. Stereological measurements showed that PVM makes up 23% of the mesophyll volume in nodulated soybean. To get an indication of the extent of involvement of PVM in ureide metabolism, physical characteristics, distribution of allantoinase activity and ureide content were determined in isolated PVM protoplasts (PVMP) and mesophyll protoplasts (MP). PVMP were larger and contained less chlorophyll and protein than MP. PVMP had twice as much allantoinase activity per protoplast but only half as much allantoinase activity when expressed on a volume basis as compared to MP. Total leaf ureide concentration was high and nearly equally distributed between MP and PVMP. PVMP had a higher ureide content per protoplast, a higher concentration of allantoic acid and a lower ratio of allantoin to allantoic acid. These results suggest that both tissues have the capacity to assimilate allantoin in vivo. The data are discussed with reference to the relative access of the two mesophyll tissues to incoming ureides.     

Ontogenetic Changes in the Level of Ureides and Enzymes of their Metabolism in Various Plant Parts of Pigeonpea (Cajanus cajan)

Levels of allantoin and allantoic acid in shoots, roots, nodulesand leaves of pigeonpea plant, in general, followed the patternof acetylene reduction in nodules, increasing progressivelyfrom 15 days after sowing (DAS) and attaining peaks at 75 DASand 60 DAS, respectively, except in shoots where their contentsevinced maximum values at pod-setting (90 DAS). Activity ofGS in nodules and shoots reached a maximum at 60 DAS and 75DAS, respectively. However, in leaves and roots, the enzymeshowed a biphasic behaviour with peaks at days 60 and 105 inleaves and at days 75 and 105 in roots. GDH activity in nodulespeaked at 60 DAS, whereas, in leaves and roots, the maximumactivity was observed at flowering (75 DAS). Uricase was presentonly in nodules with peak activity at flowering. Allantoinaseactivity again peaked at flowering, where nodules had maximumactivity followed by leaves, roots and shoots. Urease couldbe detected in all the organs with maximum activity at 60 DASin leaves followed by roots and nodules. Except uricase, allthe enzymes reported above were also present in reproductivestructures. Compared to GS, GDH was more active both in flowerbuds and developing pods. Seeds, compared to podwalls, containedhigher activities of GDH, allantoinase and urease at day 105.Only allantoin could be detected in seeds and podwalls at day105. Key words: Cajanus cajan, Allantoin, Allantoic acid, Nitrogenase, Glutamine synthetase, Glutamate dehydrogenase, Uricase, Allantoinase, Urease, Development