A spectrophotometric assay for asparaginase ...

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Aspartate formed by the action of asparaginase (obtained from corn endosperm ... tissue the asparaginase assay is prerun for a determined period of time before ...
A spectrophotometric assay for asparaginase obtained from corn (Zea mays) endosperm tissue

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SANTOSH MISRAAND A N NOAKS' Del~crr/tnen/r!f Biology, McMnsler Ur~iciersi/y,hot nil lot^, On!., Cnncrclcr L8S 4Kl Received February 7, 1980 MISRA,S., and A. OAKS.1980. A spectrophotometric assay for asparaginase obtained from corn (Zeo rnrrys) endosperm tissue. Can. J. Bot. 58: 2481-2483. Aspartate formed by the action of asparaginase (obtained from corn endosperm tissue) is converted to oxaloacetate (OAA) in the presence of a-ketoglutarate and a commercial glutamate-oxaloacetate transaminase (GOT). The OAA is then reduced to malate in the presence o f a commercial malate dehydrogenase (MDH) and NADH H+. The oxidation of NADH H+ can be followed spectrophotometrically. Since the asparaginase activity is relatively low in plant tissue the asparaginase assay is prerun for a determined period of time before GOT and MDH are added to the system. The nanomoles of NADH oxidized in the above assay are quantitatively similar to the production of aspartate and NH4+ as assayed by traditional methods. Values for 1 h are in the order of 290 nmolmg protein or 349 nmol.endosperm'.

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MISRA,S., et A. OAKS. 1980. A spectrophotometric assay for asparaginase obtained from corn (Zrtr n~trys)endosperm tissue. 1980. Can. J. Bot. 58: 2481-2483. L'aspartate forme par I'action de I'asparaginase (obtenue de I'endosperme du m a ~ sest ) transforme en oxaloacetate (OAA) en presence de a-cetoglutarate et d'une glutamate-oxaloacetate transaminase (GOT) commerciale. L'OAA est ensuite reduit en malate en presence d'une malate dishydrogenase (MDH) commerciale et de NADH H+. L'oxydation de NADH H+ peut 6tre suivie par spectrophotometrie. Puisque I'activite asparaginasique est relativement faible dans les tissus vegitaux, on laisse agir d'abord I'asparaginase pendant une piriode determinee avant d'ajouter la GOT et la MDH au systeme. Les nanomoles de NADH oxydees au cours de ce test sont quantitativement tquivalentes a la production d'aspartate et de NH4+ qui est testee par les methodes traditionnelles. Les valeurs pour I h sont de I'ordre de 290 nmol.mg proteine-I ou de 349 nmol.endosperme-I. [Traduit par le journal]

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Introduction L-Asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) is widely distributed in animals, microbes, and plant tissues and has received considerable attention in the past due to its antineoplastic activity (1 1). Asparagine, a major transport compound in legumes and cereals, is presumably a precursor for the amino acids found in storage proteins

(3' 6)' It up 70% of the transported lupin seeds only 7-10% of the residues of the seed protein is asparagine (1). Therefore deamidation of asparagine in lupin by asparaginase probably provides an important means of liberating nitrogen required for the synthesis of other amino acids. Asparginase activity can be measured by determining the production of ammonia or aspartic acid (1 1). ~ ~ ~ ofammonia i ~ by direct ~ ~t i~ ~ ~ has been in tissues (5, 10). In bacteria and animal tissue where levels of asparaginase can be quite high (50-500 'Research supported by an operating grant from Agriculture Canada. ,Author to whom correspondence should be addressed.

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nmol).min-'. mg protein-' a direct spectrophotometric procedure has also been used (2, 4). We have adapted this method for use in cereal endoSperm tissue. The method is sensitive, rapid, and quantitatively comparable to more traditional methods. Materials and methods An inbred line of corn, W64A, was used in all experiments. Endosperms from developing seeds were harvested 20 days after pollination, frozen in liquid N,, and stored at -20°C. The tissue was homogenized in 25 mM NaHEPES, pH 8.0, 12.5 mM 2-mercaptoethanol, 1 mM EDTA, and 20 mM KCI. TWOmillilitres of buffer was used per gram fresh weight of tissue. The homogenate was centrifuged at 12000g for 20 min after passing it through a single layer of Miracloth. The supernatant was brought to 50% saturation with (NH4),S04. After 20 min on ice the precipitate was redissolved in a minimum volume of elution buffer. The elution buffer had the same composition ~ ~ as the~ extraction l buffer ~ except ~ that iKC1 was ~ omitted ~ when the effect of KC1 was to be studied. The extract was layered on to a G-75 Sephadex column (1.5 cm x 20cm) which had previously been equilibrated before with elution buffer (I00 mL). The fractions containing protein were collected and pooled. The enzyme was assayed using NaHEPES (25 mM, pH 8.0) asparagine 100 mM, KC1 20 mM, and 0.2 mL of enzyme in a total volume of 0.8 mL. Before use in this assay the asparagine was

0008-4026/80/232481-03$01.OO/O @I980 National Research Council of CanadaIConseil national de recherches du Canada

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CAN. J . BOT. VOL. 58. 1980

passed over a Dowex-l (acetate) column to remove contaminating aspartic acid. The reaction mixture was incubated at 30°C for periods up to 2 h. The reaction was stopped by placing the tubes in boiling water for 5 min. The precipitated protein was separated by centrifugation in a clinical centrifuge. The supernatant was assayed for total aspartate by a coupled reaction involving glutamate-oxaloacetate transaminase (GOT) and malate dehydrogenase (MDH). The assay included 0.1 mL of NADH (0.16 mM), 0.1 mL of a-ketoglutarate (a-kg) (1 mM), GOT and MDH, and an appropriate aliquot of supernatant in a total volume of 1 mL. The total oxidation of NADH was followed at 340 nm with a Gilford spectrophotometer. The production of aspartate was confirmed by descending paper chromatography in butanol - acetic acid - water (12:3:5) or phenol, H 2 0 (80:20, w/v) solvent systems. An analysis with a Beckman model amino acid analyzer also confirmed the presence and amount of aspartate. Ammonia produced in the reaction mixture was assayed in Conway dishes. The NH,+ was liberated from the reaction mixture by adding a saturated solution of potassium carbonate (3 mL/mL of reaction mixture). It was allowed to distill into 0.0143 N sulfuric acid. the sulfuric acid sample was then assayed for NH,+ by the phenolhypochlorite reaction (7). GOT, MDH, and NADH were obtained from Sigma Chemical Co. and asparagine was purchased from Calbiochem.

Results and discussion In the reaction sequence used in this technique, asparagine is first hydrolyzed by asparaginase to aspartic acid and ammonia (reaction 1). Aspartic acid is then converted to oxaloacetate (OAA) by GOT (reaction 2) and OAA is then reduced to malate by MDH in the presence of NADH (reaction 3).

[2] [3]

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+ H 2 0 Asnase GOT L-Asp + a-kg OAA + NADH + H+

[l] L-Asn

L-Asp

NH,

L-Glu

OAA

MDH

Malate

TABLE1. Requirements for the assay of asparaginase in extracts prepared from maize endosperm (W64A) 20 days postpollination Activity, nmol NADH/assay

Assay conditions Preincubation mixture" Complete Complete minus KC1 Complete 0 time Minus enzyme Minus Asn Coupled reactionh Complete plus AOA (1 niM) Minus transaminase minus M D H Minus Asn plus a-kg plus NADH

No activity No activity 0.2

aThe complete preincubation mixture contained 100 m M Asn, 20 m M KCI, 25 m M NaHEPES (pH 8.0), 0.2 mL enzyme in a total volume o f 0.8 mL. bThe coupled reaction contained supernatant from preincubation mixture (see Materials and methods); 0.1 mL o f N A D H (0.16 mM), 0.1 mL a-kg (I mM),MDH(1.27pg protein.assay - I ) , and GOT (20 pg prote~n.assay- I ) .

TABLE2. Stoichiometry of the asparaginase reaction" Time of incubation, min

NADH, nmol/assay

Asph nmol/assay

NH,+, nmol/assay

0 20 40 60 80 80 ( - A m )

10.68 46.8 83.6 116.8 143.6 0-0.8

21.6 45.6 87.6,84.4 116.4, 114.8 162 0.08

17.2 54.0 95.09 124.58 155.44 Not detectable

OComplete reaction as described in Table 1 . bAspartate levels were determined by amino acid analyzer. At 60 min the level o f Asp was 349 nmol.endosperm - and 290 nmol.mg protein - I .

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The total decrease in concentration of NADH followed at 340 nm should equal the aspartic concentration of the reaction mixture, if there are no interfering reactions. In our system a calibration curve was prepared using various concentrations of aspartic acid and the reaction was then tested with Sephadex-treated enzyme. The reaction requires asparagine, endosperm extract, GOT, and MDH (Table 1). The reaction was enhanced by KC1 and hence is similar to the asparaginase system described for legume cotyledons (8, 9). KC1 was also required during the extraction and elution procedures to ensure maximum activities. The addition of a-amino oxyacetate (AOA) to the coupled reaction assay system completely inhibited the oxidation of NADH. With our assay conditions maximum activity was obtained at pH 8.0. The activity was linear with time for the initial 80 min and was proportional to the amount of enzyme added. Asparagine at 100 mMand KC1 at 20 mM saturated the reaction. After chromatography of the reaction

mixture minus GOT and MDH a ninhydrin positive spot with an R fsimilar to aspartate was detected. More aspartate was present with increasing incubation time and no aspartate was detected when either asparagine or enzyme was omitted from the reaction. This observation was confirmed when the products were quantitatively estimated with an amino acid analyzer (Table 2). There is a good correlation in the levels of aspartate formed whether aspartate levels were determined by the oxidation of NADH or by direct analysis. The direct assay of NH,+ also yields results predicted by the stoichiometry of the reaction. This observation plus the lack of oxidation of NADH in the minus asparagine treatment show that there is no interference from glutamate dehydrogenase and other NADH consuming reactions, and that the method is therefore a reliable assay for asparaginase activity. The method can also be adapted for the measurement of asparagine and aspartate levels in the tissue. Under optimum assay conditions asparaginase activity per endosperm was 0.349 pmoles - h-'. At

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MlSRA A N D OAKS

20 days postpollination levels of asparaginase have reached their highest values in the developing endosperm (Misra et al., in preparation). Activities reaching 1.2 pmol . h-' . seed-' in developing pea cotyledons and 3.6 pmol. h-I. seed-' in the testa have been reported (9). Thus, cereal endosperm appears to have much less asparaginase than legume cotyledons. The relatively lower level of activity in corn endosperm tissue suggests that this pathway may not be as important in the supply of nitrogen for general assimilation in corn seeds as it is in legumes. Acknowledgements We would like to thank Dr. Pierre Gadal for many discussions in the early phases of this work and Dr. K. W. Joy for assaying aspartate level with his amino acid analyzer (Table 2). 1. ATKINS,C. A., J . S. PATE,and P. J. SHARKEY. 1975. Asparagine metabolism; key to the nitrogen nutrition of developing legume seeds. Plant Physio1.56: 807-812. 2. COONEY, D. A., and R. E. HANDSCHUMACHER. 1968. Investigation of L-asparagine metabolism in animals and human subjects. Proc. Am. Assoc. Cancer Res. 9: 15.

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3. IVANKO, S., and J . INGVERSEN. 1971. Investigation on the assimilation of nitrogen by maize roots and the transport of some major nitrogen compounds by xylem sap. Physiol. Plant. 24: 355-362. 4. KOJIMA,Y., and W. E. C. WACKER.1969. An enzymatic method for the measurement of asparagine and a new assay for asparaginase activity. J. Lab. Clin. Med. 74: 521-526. 5. MEISTER,A. 1955. L-Asparaginase from Guinea pig serum. Itz Methods in enzymology. Vol. 2. Edited by S . P. Colowick and M. 0 . Kaplan. Academic Press Inc. New York. p. 383. 6. PATE,J. S. 1973. Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem. 5: 109-1 19. 7. RUSSEL,J. A. 1964. A colorimetric estimation for small amounts of ammonia by phenyl-hypochlorite reaction. J. Biol. Chem. 156: 457-461. 8. SODEK, L., P. J. LEA,and B. J. MIFLIN.1978. A potassium dependent asparaginase from maturing legume and cereal seeds. Plant Physiol. Suppl. 61: 378. 9. SODEK,L., P. J. LEA, and B. J. MIFLIN. 1979. Asparaginase activity in developing seeds of Pislrrn sotiulrm. Plant Physiol. 63: (Supp. 5). 142. J. C., JR. 1970. L-Asparaginase. In Methods in 10. WRISTON, enzymology. Vol. 17A. Edited by S. P. Colowick and M. 0 . Kaplan. Academic Press Inc., New York, London. pp. 732-742. 11. WRISTON, J. C., and T. 0 . YELLIN.1973. L-Asparaginase,a review. In Advances in enzymology. Vol. 39. Edited by A. Meister. Interscience, New York, NY. pp. 185-245.