Solubilization and extraction of allantoinase and allantoicase from the green alga <i>Chlamydomonas reinhardtii</i>

Piedras, Pedro; Cárdenas, Jacobo; Pineda, Manuel

doi:10.1002/pca.2800060503

Cited by 12 publications

(5 citation statements)

References 20 publications

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“…The inclusion of detergent could stabilize hydrophobic domains within the enzyme as well. DOC has been used to solubilize other allantoinases (Costigan et al 1987, Ory et al 1969, Piedras et al 1995, Thomas et al 1983). The total allantoinase activity was purified 200-fold, suggesting that allantoinase might represent 0.5% of the total protein present in French bean developing fruits, assuming that activity does not change during the purification process.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots were taken at different times and 0.2 ml of 0.15 M HCl and water at 4°C were added to a final volume of 1 ml. Samples were boiled for 10 min and the glyoxylate formed was determined as previously described (Piedras et al 1995). One unit of enzyme is defined as the amount that catalyses the formation of 1 μmol of glyoxylate per minute.…”

Section: Methodsmentioning

confidence: 99%

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Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris)

Raso

Pineda

Piedras

2007

Physiologia Plantarum

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Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the hydrolysis of allantoin to allantoic acid, a key reaction in the biosynthesis and degradation of ureides. This activity was determined in different tissues of French bean plants (Phaseolus vulgaris L.) which were grown under nitrogen-fixing conditions. Allantoinase activity was detected in all tissues analysed, but the highest levels of specific activity were found in developing fruits, from which allantoinase has been purified to electrophoretic homogeneity and further characterized. After diethylaminoethyl (DEAE)-Sephacel chromatography, two peaks showing allantoinase activity were obtained in the chromatographic profile and the corresponding proteins were independently purified. Total allantoinase activity was purified 200-fold, indicating the relevance of this enzymatic activity in French bean developing fruits, with allantoinase representing 0.5% of total soluble protein. Both proteins with allantoinase activity are monomeric with molecular masses of 45 and 42 kDa. The specific activities of the purified proteins were 560 and 295 units mg(-1), which correspond to turnover numbers of 25,200 and 12,100 min(-1), respectively. The two proteins have very similar biochemical properties showing Michaelis-Menten kinetics for allantoin with K(m) values of about 60 mM, with high optimal temperatures; are metalloenzymes; are inhibited by compounds reacting with sulphydryl groups; and are unaffected by reducing agents. All analysed tissues exhibited the two activities responsible for allantoin degradation, although one of them was the main form in leaves (the most photosynthetic tissue) and the other protein was the main form in roots (non-photosynthetic tissue). The allantoinase activity and distribution of both proteins have been analysed during fruit development. For both proteins, the allantoinase activity and distribution pattern were the same in plants growing either under nitrogen-fixing conditions or fertilized with nitrate.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris)

Raso

Pineda

Piedras

2007

Physiologia Plantarum

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“…Allantoate, ureidoglycolate and glyoxylate were determined as described by Piedras et al (1995). A value of 42,368 M ¡1 cm ¡1 was calculated for the molar extinction coeYcient of diphenilformazan.…”

Section: Analytical Determinationsmentioning

confidence: 99%

Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine

Raso

Muñoz

Pineda

et al. 2007

Planta

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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 degrees 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.

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“…The high value of K m (about 250 mM) determined for mouse allantoicase renders it very improbable that the enzyme is effective under in vivo conditions. This hypothesis is further supported by the evidence that the functional allantoicases characterized so far (from microorganisms to fishes) possess K m values ranging from 4 to 9 mM [27–29].…”

Section: Discussionmentioning

confidence: 56%

Property comparison of recombinant amphibian and mammalian allantoicases

et al. 2002

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Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional allantoicase, therefore we expressed the Xenopus and mouse allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme. ß

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Solubilization and extraction of allantoinase and allantoicase from the green alga Chlamydomonas reinhardtii

Cited by 12 publications

References 20 publications

Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris)

Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris)

Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine

Property comparison of recombinant amphibian and mammalian allantoicases

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