Carboxypeptidase Activity in Poppy Seedlings, Papaver somniferum L.

J.PlantPhysiol. Vol.143.pp.161-164{1994}

Carboxypeptidase Activity in Poppy Seedlings, Papaver somniferum L. MARIA BENEsovA., PETER KovA.cs, and MxKur.As PsENAK Department of Cell and Molecular Biology of Drugs Faculty of Pharmacy of Comenius University, 832 32 Bratislava, Slovakia Received June 26, 1992 ·Accepted October 1, 1993

Summary

The developmental profile and distribution of carboxypeptidase activity in developing poppy seedling and in its organs was determined. Carboxypeptidase activity exhibited two pH optima: at pH 7.5 and at pH 5.0. The enzyme activity with pH optimum at pH 7.5 increased 4-fold and that with pH optima at pH 5.0 increased by a factor 2.6 during poppy seedlings development. Two active fractions (I and II) were obtained by protein separation (fraction obtained between 50-80% ammonium sulphate precipitation) on Sephadex G-150. By further separation of protein in fraction II on DE-22 cellulose two active fractions (CP1 and CP2) were obtained. CP, appeared to be a serine type enzyme and CP2 is probably a metalloenzyme. In fraction CP1 and CP2 different forms of carboxypeptidase were identified by PAGE. These results indicate the presence of multiple forms of carboxypeptidase in developing poppy seedlings.

Key words: Carboxypeptidase, purification and properties, Papaver somniferum L. seedlings. Abbreviations: Bz = benzoyl; CP = carboxypeptidase; DFP = diisopropylfluorophosphate; PMSF phenylmethane sulphonyl fluoride; pCMB = p-chloromercuribenzoate. Introduction

Poppy seeds belong to a group of oil seeds. Their content of protein is around 25 % on dry weight basis (Eklund and Agren, 1973). The aleurone grains are localized in the outer layer of endosperm and in significant lesser extent within the cells of the embryo (Sarkany et al., 1976; Psenak et al., 1985). Proteolytic activities are essential for the mobilization and reutilization of protein reserves during seed germination (Preston and Kruger, 1976). The principal alkaloid of poppy seedling is thebaine (Wieczorek et al., 1986) which starts to form immediately after radicle protrussion (Psenak et al., 1987). Tyrosine is the basic precursor of poppy alkaloids (Loeffler et al., 1987). Tyrosine used by developing poppy seedling for thebaine biosynthesis may in part be formed from reserve proteins. In our previous paper aminopeptidase (Benesova et al., 1974, 1980, 1984; Kovacs and Benesova, 1976), dipeptidase (Kovacs, 1976), dipeptidylpeptidase IV (Benesova et al., 1987) and proteinase (BAPAse) (Kovacs et al., 1985) from © 1994 by Gustav Fischer Verlag, Stuttgart

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poppy seeds and developing poppy seedlings were characterized. In this paper results are presented indicating the multiple forms of carboxypeptidase in poppy seedlings.

Materials and Methods

Plant material

Seeds of opium poppy (Papaver somniferum L. cv. Amarfn) were germinated in Petri dishes (in the dark, 25 °C, relat. humidity 75 %). Dry seeds (0.3 g) were uniformely distributed on a dish moistened with 80 mL of distilled water. The endosperm and embryos and latter on hypocotyls, cotyledones and roots were separated using a magnifier. The plant parts were immediately inserted into a homogenisation buffer and kept at 4 oe, Enzyme preparations

For enzyme extraction 100 germinating seeds or isolated organs of poppy seedlings were homogenized in the presence of 10 mg of

162

MAluA. Bi!NEWv.A, PETER Kov.Acs, and Mntm.As PSEN.AK

Polyclar AT (Serva) (at 0 °C in glass homogenizers in 1 mL of 0.2 molL - 1 of Tris-HCI buffer, pH 7.2). After centrifugation (0 °C, 250,00 x g, 20 min) the supernatant obtained was used as the initial enzyme preparation. The purification of carboxypeptidase started with 100 mL of the initial enzyme extract prepared from 4-day-old seedlings. After ammonium sulphate saturation the sediments obtained by centrifugation (as above) was dissolved in minimum amounts of homogenisation buffer and dialyzed overnight against a 10-fold volume of 0.01 molL - 1 of Tris-HCl (pH 7.2) at 4 °C. The protein fraction between the 50-80% ammonium sulphate saturation was further purified on a Sephadex G-150 column (1.8 x 50 em) equilibrated with 0.02molL - 1 of the Tris-HCl buffer (pH 7.2). Fractions (3 mL) were collected at a flow rate of 15mL·h- 1 using Tris-HCl buffer for the elution of proteins. The fractions with highest activity (N° 49-53) were joined and further purified on a DE-22 dimethylaminoethylcellulose (Whatman) column (1.5x25cm) equilibrated with 0.05molL- 1 of K+phosphate buffer (pH 7.2). Protein was eluted with a linear gradient of K+-phosphate buffer, pH 7.2. The amount of protein was determined according to Lowry et al. (1951) or spectrophotometrically at 260 and 280nm (Bergmeyer, 1970).

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Fig.1: The pH-profile of carboxypeptidase activity. Buffer solutions: 0.05 mol· L - 1 - - 0 - - acetate, --£::.-- K+ -phosphate, - 0 - - Tris-HCl. Substrate: B.-Gly-Phe, 37 °C.

Determination ofthe carboxypeptidase activity The carboxypeptidase activity was determined as described by Visuri et al. (1969). Benzoylglycylphenylalanine (B.-Gly-Phe) was used as substrate. The reaction mixture contained in a fmal volume of 0.4mL: 3011L of substrate (3mmoiL- 1), 0.02molL- 1 of TrisHCl (pH 7.2). The reaction was initiated by addition of enzyme (50 11L) at 37 °C. The amount of released phenylalanine was determined by ninhydrine method at 570 nm. In control samples inactivated enzyme ( 10 min, 100 °C) solution was used. One unit of carboxypeptidase activity was defmed as the liberation of 111mol phenylalanine/min at 37 °C.

Characterization ofthe carboxypeptidase The pH-profile of carboxypeptidase activity was determined within a pH range from pH 3.5 to pH 8.5: 0.05molL - 1 of acetate, K+-phosphate and Tris-HCl buffers, respectively, were used. In these experiments the initial enzyme solution obtained from 36-hrs seedlings was used. For testing the effect of DFP (diisopropylfluorophosphate), PMSF (phenylmethane sulphonyl fluoride), 1.10 - phenantroline (solution in 0.2molL - 1 of Tris-HCl buffer pH 7.2) and iodoacetic acid (solution in distilled water) and enzyme preparation with specific activity of 121 pkat · mg proteins - 1 was used.

Electrophoresis on polyacrylamide gels Disc electrophoresis was carried out as described by Doi et al. {1980 b) in 5% polyacrylamide gels, of pH 5.0 (3mA per tube, 2 hours at 4 °C). Gels were incubated with B.-Gly-Phe at pH 5.0 (for CP1) and at pH 7.5 (for CP2) and (37°C for 10min); subsequently the gels were transferred into a 1 % ninhydrine solution containing 50% of methyl cellosolve, 0.1 molL - 1 of citrate buffer (pH 5.2) and 0.1% of SnCh. The pertinent bands of carboxypeptidase activity were visualised by heating the gels 20 min.

Results and discussion

Under the used experimental conditions the radicule protrusion and the growth and development of poppy seedling

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Fig. 2: Developmental profile of carboxypeptidase in the individual organs of poppy seedlings: - - 0 - - seeds or whole seedlings, - - x - - endosperm,--£::.-- embryo (cotyledons+ hypocotyl), - 0 - - roots, pH 7.5;---- -0----- seeds or whole seedlings, pH 5.0; substrate B.-Gly-Phe, 37 °C.

are accomplished up to 24 h and between the 1st and 3rd days post imbibition, respectively (Psenak et al., 1985}. First the pH-profile of carboxypeptidase activity was tested. An initial enzyme preparation from 36-hrs old seedlings free from endosperm was used. The enzyme exhibited two pH optima, at pH 5.0 and 7.5 (Fig. 1}. The pH-optima of plant carboxypeptidases are usually between 3.5 and 5.5 (Ryan and Walker-Simmons, 1981}. A carboxypeptidase activity with pH-optimum of pH 7 has been reported in rice seedlings (Doi et al., 1980 a, c) and in the scutella of maize seedlings (Mikola and Saarinen, 1986}. The developmental changes of the enzyme activity in whole poppy seedlings and in their individual organs are shown on Fig. 2. Using initial enzyme preparations from whole poppy seedlings an increase in enzyme activity was

Carboxypeptidase in Poppy Seedling

163

Table 1: Purification of carboxypeptidase from poppy seedlings. Substrate used: B.-Gly-Phe, pH 7.2 at 37 °C. Enzyme fraction

Volume (mL)

Protein (mg · mL - 1)

Activity (pkat · mL - 1)

(NH4)2S04 precipitation 50-80%

8

4.1

18.4

4.5

Gel filtration on Sephadex G-150 fractions (41-44), (form I) fractions (49- 53), (form II)

9 9.1

0.5 0.4

22.1 30.8

44.2 75.1

9.9 16.7

Chromatography on DE-22 cellulose CP1-fraction 18 CPrfraction 29

3 3.2

0.06 0.08

18.4 9.7

306.7 121.3

68.2 27

Specific activity (pkat · mg P- 1)

Purification (fold)

P - protein observed by a factor of 4 at pH 7.5 and by a factor of 2.6 at pH 5. Similar results were reported for carboxypeptidase in developing rice seedlings (Doi et al., 1980 b). A noteworthy enzyme activity (measured at pH 7.5) is present in endosperm at the very beginning of poppy seedlings growth and development implying the early mobilization of endosperm protein reserves. An inverse relationship between carboxypeptidase activity in endosperm and in cotyledones indicates that the seed's reserve redistribution (after partial hydroly-

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sis) is a significant event in their reutilization during the growth and development of poppy seedlings. The protocol adopted for the preparation of a partially purified carboxypeptidase from 3-day-old endosperm free poppy seedlings is summarized in Table 1. Two active forms (I and II) have been obtained by protein. separation on a Sephadex G-150 column. Fractions No 49-53 corresponding to activity peak II were joined and further purified on a column of DE-22-cellulose. Two active fractions (CPt. CP2) have been obtained (Fig. 3). Fraction CPt and CP2 have been used to test the effect of inhibitors, for CP1 at pH 5.0 and for CP2 at pH 7.5 (Table 2). PMSF and DEP produced almost complete inhibition of CP1. Sulfhydryl reagents and metalloenzyme inhibitors did not affect the enzyme activity. The findings suggest that

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Fig. 3: Ion exchange chromatography of carboxypeptidase on a DE 22 cellulose column.----- protein,-- carboxypeptidase activity. Substrate: B.-Gly-Phe. Table 2: Effect of inhibitors on partially purified carboxypeptidase fractions (CP 1 and CP2) from poppy seedlings. The enzymes (CP, = SO ~-tg protein and CP2 = 41~-tg protein) were preincubated in 0.5 mL 0.05 mol · L -I acetate buffer (pH 5.0) for CP 1 and Tris-HCl (pH 7.2) for CP2 containing various inhibitors for 10 min. After preincubation B.-Gly-Phe was added and activity was assayed. Inhibitors None PMSF

DFP

Iodoacetic acid Iodoacetamide pCMB HgCh EDTA 1.10-Phenantroline 2-Mercaptoethanol

Concentration (mmol· L- 1) 0.1 0.1 2.0 2.0 0.1 0.1 2.0 2.0 1.0

Remaining CPt

activity(%) CP2

100 15

100 96 98 65 45 16 18 10 15 10

11

89 86 84 80 98 93 100

Fig. 4: Disc electrophoresis of carboxypeptidase CP1 and CP2 from poppy seedlings. Cationic run at pH 5.0 in 5% polyacrylamide gel (20 jlg proteins, 3 rnA per tube, 2 hrs). Bands were visualised with ninhydrine reagent after enzyme reaction with B.-Gly-Phe.

164

MARIA BENJ!Sov.A, PETER KovA.cs, and Mntur.As PsENAK.

CP1 might be a serine type carboxypeptidase. Carboxypeptidase from higher plants with pH-optima in acidic region has been reported to be serine carboxypeptidase (Ryan and Walker-Simmons, 1981). Fraction CP2 appeared to be sensitive to sulfhydryl reagents {Table 2) and metalloenzyme inhibitors implying that the SH groups and a metal are essential for this carboxypeptidase activity. Thiols such as dithiothreitol or mercaptoethanol deactivate copper - containing enzymes (Harris et al., 1974; Stafford, 1975). Mercaptoethanol {1 mmolL -I) produced almost complete inhibition of CP2. This indicates that CP2 might be a copper metalloenzyme. An enzyme with similar properties was described by Doi {1980 c) in rice seedling. By disc electrophoresis different forms of carboxypeptidase were identified in fraction CP1 and CP2{Fig. 4). These results suggest the occurrence of multiple forms of carboxypeptidase in developing poppy seedlings. References BENI!Sov.A, M., P. KovA.cs, and K. SENKPIEL: Aminopeptidaseaktivitat in Mohnkeimlingen (Papaver somniferum L. cv. Dubsky). Biochem. Physiol. Pflanzen 165, 173 -179 (1974). BENESbv.A, M., K. SENKPIEL, P. KovA.cs, and A. BARTH: Multiple molekulare Aminopeptidaseformen in Mohnkeimlingen (Papaver somniferum L. cv. Dubsky). Biochem. Physiol. Pflanzen 175, 252-262 {1980). BENE5ov.A, M., P. KovA.cs, and A. BARTH: Effect of amino acids and peptides on L-Ala-aminopeptidase activity in germinating poppy plants (Papaver somniferum L.). Biol6gia (Bratislava) 39, 769775 {1984). BENI!Sov.A, M., P. KovAcs, M. P5ENAK., and A. BARTH: Dipeptidylpeptidase of poppy seedlings (Papaver somniferum L. cv. Amarin). Biol6gia (Bratislava) 42, 779-789 {1987). BEllGMEYER, H. U.: Methoden der enzymatischen Analyse, Band I, p. 139, Akademie-Verlag, Berlin {1970). Dor, E., N. KoMORI, T. MATOBA, and Y. MoRITA: Some properties of carboxypeptidases in germinating rice seeds and rice leaves. Agric. Biol. Chern. 44,77-83 (1980 a). - - - - Purification and some properties of carboxypeptidase in rice bran. Agric. Biol. Chern. 44, 85-92 {1980 b). - - - - Carboxypeptidase-like metal enzyme in rice seedlings. Agric. Biol. Chern. 44, 921-922 (1980 c). EKLUND, A. and G. AGREN: Nutritive values of poppy-seed protein. Abstract book 9th International Congr. Biochem. Stockholm, M-19, p. 463 (1973). HARRis, E. D., W. A. GoNNEllMAN, J. E. SAVAGE, and B. L. 6oELL: Connective tissue amine oxidase II. Purification and partial char-

acterization of lysyl oxidase from chick aorta. Biochim. Biophys. Acta341, 332-344 {1974). KovAcs, P. and M. BENE5ovA: Some properties of aminopeptidases from seedlings of Papaver somniferum L. cv. Dubsky. Biol6gia (Bratislava) 31, 423-430 {1976). KovAcs, P.: Dipeptidase activity in Papaver somniferum L. cv. Dubsky seedlings. Biochem. Physiol. Pflanzen 169, 273-279 {1976). KovAcs, P., J. DUFKOVA-BARANcmwvA, and M. PsENAK.: Proteinase and N-benzoyl-DL-arginine-p-nitroanilide hydrolase in poppy seedlings (Papaver somniferum L.). Biol6gia (Bratislava) 40, 345-356 (1985). LoEFFLER, S., S. STADLE!l, N. NAGAKURA, and M. H. ZENK: Norcoclaurine as biosynthetic precursor of thebaine and morphine. J. Chern. Soc. Commun. 1160-1162 {1987). LoWRY, 0. H., N.J. RosEBROUGH, A. L. FAllll, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. Biol. Chern. 193, 266-275 {1951). MIKOLA, L. and S. SAARINEN: Occurrence of acid and neutral carboxypeptidase in germinating cereals. Physiol. Plant. 67, 557-561 {1986). PllESTON, K. R. andJ. E. KlluGEll: Purification and properties of two proteolytic enzymes with carboxypeptidase activity in germinated wheat. Plant Physiol. 58, 516-520 (1976). PsENAK., M., P. KovAcs, T. LisZT, S. SAilKANY, and L. FRIDVALSKY: Biochemical and histochemical studies of seeds and seedlings of Papaver bracteatum Lindl. Acta Facult Pharm Univ Comen 39, 89-112 (1985). PsENAK., M., H. SMOGllOVIcovA, L. BEZI.KovA, and P. KovA.cs: Rast a respiracia kli~nych rastlfn maku vo vzt'ahu k tvorbe tebafnu. VII. Ceskoslov. farm. 36, 416-419 (1987). RYAN, C. A. and M. WALKEP.-SIMMONS: Plant proteinases. In: STUMPF, P. K. and E. E. CoNN (eds.): The Biochemistry of Plants, Vol. 6, p. 321-350, Acad. Press, New York {1981). SAilKANY, S., C. MlcHELS-NYoMAli.KAY, and J. SAilKANY-Ktss: Accumulation of alkaloids in Papaver somniferum L. In: LucKNE!l, M., K. MoTHES, and L. NoVEll (eds.): Secondary Metabolism and Coevolution. Deutsche Akademie der Naturforscher Leopoldina, Halle (Saale), p. 83-88 (1976). STAFFORD, H.: Transformation of a large aggregate of hydroxycinnamate hydroxylase to lower molecular weight forms by sulfhydryl agents in green leaves of Sorghum. Plant Physiol. 55, 145149 (1975). UMETsu, H. and E. lcHISHIMA: Action on peptides by wheat carboxypeptidase. Phytochemistry 22, 591-592 {1983). VIsURI, K., J. MIKoLA, and T. M. ENARI: Isolation and partial characterization of a carboxypeptidase from barley. Eur. J. Biochem. 7, 193-199 (1969). WmczollE!l, U., N. NAGAKURA, CH. SUND, S. JENDllZEJEWSKI, and M. H. ZENK: Radioimmunoassay determination of six opium alkaloids and its application to plant screening. Phytochemistry 25, 2639-2646 {1986).