Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Biochimica et Biophysica Acta 1631 (2003) 153 – 159 www.bba-direct.com

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.) Marek Oblozinsky a,b, Regina Schoeps b, Renate Ulbrich-Hofmann b,*, Lydia Bezakova a a

Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, SK-83232 Bratislava, Slovak Republic b Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany Received 22 January 2002; received in revised form 9 September 2002; accepted 27 November 2002

Abstract Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazu´r (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca2 + ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn2 + ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase D; Papaver somniferum; Purification; Zn-activation; Transphosphatidylation

1. Introduction Phospholipase D (phosphatidylcholine choline hydrolase, PLD, EC 3.1.4.4) is an important enzyme in phospholipid metabolism, which is widely distributed in plant kingdom [1,2]. In poppy (Papaver somniferum L., Papaveraceae), however, it has been not described so far. PLD catalyses the hydrolysis of glycerophospholipids such as phosphatidylcholine (PC) to form phosphatidic acid (PA) and the free polar head group of the phospholipid. Recently, PLD has

Abbreviations: ECL, enhanced chemiluminescence; EDTA, ethylenediamine tetraacetic acid; HMW, high molecular weight; HPTLC, high performance thin layer chromatography; IEF, isoelectric focusing; JA, jasmonic acid; MES, morpholinoethansulfonic acid; PA, phosphatidic acid; PAGE, polyacrylamide gel electrophoresis; PC, phosphatidylcholine; PG, phosphatidylglycerol; PLD, phospholipase D; PpNP, phosphatidyl-p-nitrophenol; SDS, sodium dodecylsulfate; Tris, tris(hydroxymethyl)aminomethane * Corresponding author. Tel.: +49-345-5524864; fax: +49-3455527303. E-mail address: [email protected] (R. Ulbrich-Hofmann).

attracted much attention because of its participation in cellular regulation via transmembrane signalling [3 –5]. In general, cellular functions of PLD in plants may be divided in three general categories: lipid degradation, cellular regulation and membrane remodeling [2]. PLD is also involved in lipid-based signalling via the octadecanoid pathway [6,7], which leads to the production of regulators such as jasmonic acid (JA). JA and related oxylipins act as endogenous signals activating defence gene expression [8]. There is evidence that octadecanoid acid-derived compounds are essential elements in the regulation of secondary pathways [9]. In addition to hydrolytic activity of PLD, the enzyme also can transfer the phosphatidyl moiety to various alcohols to form the corresponding phosphatidyl alcohols. The function of this transphosphatidylation reaction in nature is still unknown, while it is widely used in biotechnology for the biocatalytic exchange of the head group in phospholipids [10,11]. Because of the distinct importance of poppy seedlings in the production of secondary metabolites, it is highly interesting whether PLD is involved in these processes. In developing poppy seedlings, alkaloids (particularly the-

1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1388-1981(02)00362-1

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baine) can be detected after few days of post-imbibition [12,13]. Hence, the lipid metabolism of poppy seedlings is important for growth and development as well as for the opium alkaloid synthesis. The present paper proves the presence of PLD in poppy seedlings. Two isoenzymes are isolated and characterized with respect to the hydrolytic and transphosphatidylation activities as well as the pH and metal ion influences on the activities. The apparent molecular masses and isoelectric points of the enzymes are determined. The presence of carbohydrate moieties is detected.

2.3. Acetone powder preparation Endosperms (2 days old) from 10.0 g of fresh poppy seedlings were ground in a mortal with a small amount of cold acetone and then homogenized in a chilled Waring blendor with 300 ml of cold acetone (with addition of about 300 g of solid CO2). The precipitate was washed and rinsed with the cold acetone until the filtrate was colorless and transparent. The residue was vacuum-dried. The resulting acetone powder was stable for several months at 4 jC. 2.4. PLD purification

2. Materials and methods 2.1. Materials Poppy seeds (P. somniferum L. cv. Lazu´r) were harvested in 2000. Phosphatidyl-p-nitrophenol (PpNP) was synthetized from L-a-lecithin from egg yolk (Fluka, Germany) according to Ref. [14]. PC (99.0%), phosphatidylglycerol (PG) (98.0%) and PA (99.3%) from soybean were a gift from Lipoid GmbH (Ludwigshafen, Germany). Octyl-sepharose CL-4B was purchased from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Water-free diethyl ether and waterfree glycerol were products of Merck (Darmstadt, Germany). High molecular weight (HMW) calibration kit for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), enhanced chemiluminescence (ECL) glycoprotein detection module and PhastGel isoelectric focusing (IEF) 3-9 were from Amersham Pharmacia Biotech. IEFmarkers liquid mix 3 – 10 was from Serva (Heidelberg, Germany). Microsep OMEGA 100 kDa membranes were from PALL GmbH (Dreieich-Sprendlingen, Germany). Nitrocellulose blotting membranes were from Sartorius AG (Go¨ttingen, Germany). High performance thin layer chromatography (HPTLC) plates (silica gel 60) were from Merck. N-glycosidase F from Flavobacterium meningosepticum was from Boehringer Mannheim GmbH (Mannheim, Germany). Bovine serum albumin was from Pierce (Rockford, USA). Peroxidase from horseradish was from SigmaAldrich Chemie GmbH (Steinheim, Germany) and aldolase was from Boehringer Mannheim. All other chemicals were products of the highest quality commercially available. 2.2. Plant material cultivation Poppy seeds were germinated on a polyurethane foam layer (thickness 10 mm) in a petri dish (2.5  18 cm) covered by a nylon cloth, using 50 ml of distilled water. The seeds (0.5 g per dish) were evenly spread on the surface. The germination process was performed in the dark at 25 jC and 70 –80% relative humidity. On the second day of post-imbibition endosperms of the seedlings were separated from the developing seedlings.

Acetone powder (2.0 g) was homogenized in 50 ml of 0.1 M sodium acetate buffer/10 mM CaCl2/6 mM cysteine hydrochloride (pH 5.5) and centrifuged at 12,000  g and 4 jC for 10 min. Extracts were treated with (NH4)2SO4 (60% saturation) and centrifuged at 26,000  g and 4 jC for 45 min. The sediment was dissolved in a minimum volume of 0.1 M sodium acetate buffer/10 mM CaCl2/6 mM cysteine hydrochloride (pH 5.5). After dialysis against 0.01 M sodium acetate buffer/50 mM CaCl2 (pH 5.5), the enzyme solution was applied onto Octyl-sepharose CL-4B column (1.0  20.0 cm). The proteins were eluted at a flow rate of 9 ml/h by a three-step elution with the following solutions: (I) 0.01 M sodium acetate buffer/50 mM CaCl2 or 50 mM ZnCl2 (pH 5.5), (II) 0.005 M sodium acetate buffer/ 30 mM CaCl2 or 30 mM ZnCl2 (pH 5.5), (III) 0.005 M sodium acetate buffer/0.1 mM ethylenediamine tetraacetic acid (EDTA) (pH 5.5). The activity was determined toward PpNP at pH 5.5 and pH 8.0 as described in Section 2.6. The united active fractions were concentrated by using 100-kDa membranes. 2.5. Protein determination In all experiments the protein content was determined according to Ref. [15] using bovine serum albumin as standard. 2.6. Hydrolytic activity in aqueous system The hydrolytic activity of PLD was determined in aqueous system by measuring p-nitrophenol released from PpNP in a procedure similar to that in Ref. [14]. The assay mixture contained 50 Al of substrate solution (10 mM PpNP, 5% (v/v) Triton X-100 and 5 mM SDS), 300 Al of buffer (0.1 M tris(hydroxymethyl)aminomethane (Tris) – HCl (pH 8.0) or 0.1 M sodium acetate buffer (pH 5.5)), 10 mM CaCl2 and 50 Al of enzyme solution. After incubation for 3 min at 30 jC, which is in the linear range of the progress curve, the reaction was stopped by addition of 100 Al of 1 M Tris – HCl/0.1 M EDTA (pH 8.0). The absorbance of the solution was determined at 405 nm and related to a standard curve for p-nitrophenol.

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For characterizing the activity of PLD-A and PLD-B, the isoenzymes were assayed at (i) different pH values in the presence of 10 mM CaCl2 (pH 4.0– 5.5: 0.1 M sodium acetate buffer; pH 6.0: 0.1 M morpholinoethansulfonic acid (MES) buffer; pH 6.5– 8.5: 0.1 M Tris – HCl buffer), (ii) in the presence of different concentrations of CaCl2 (1 –50 mM) at pH 5.5 (0.1 M sodium acetate buffer) and pH 8.0 (0.1 M Tris – HCl buffer), and (iii) in the presence of different concentrations of ZnCl2 (1 –50 mM) at pH 5.5 (0.1 M sodium acetate buffer). 2.7. Transphosphatidylation and hydrolytic activity in twophase systems Transphosphatidylation and hydrolytic reactions were performed in two-phase systems in a procedure similar to that of Ref. [16]. The reaction mixtures consisted of 620Al diethyl ether containing 1.32 Amol PC, 10 Al (136.8 Amol) of glycerol (for determination of transphosphatidylation activity) or 10 Al of water (for determination of hydrolytic activity), 30 Al of 0.1 M Tris – HCl buffer/40 mM CaCl2 (pH 8.0) or 30 Al of 0.1 M sodium acetate buffer/40 mM CaCl2 (pH 5.5) and 40 Al of purified enzyme (PLD-A or PLD-B). The reactions were performed at 30 jC in vials sealed by Teflon – silicon septa with shaking at 250 min 1. During the reaction aliquots (25 Al) of the organic phase were withdrawn and analyzed by HPTLC. One microliter of the organic solution was applied to HPTLC plates by using Desaga AS 30 TLC applicator. Phospholipids were visualized by dipping into 10% (w/v) CuSO4 in 8% (v/v) H3PO4 and drying at 170 jC. Phospholipids were evaluated with a CD 60 densitometer (Desaga) in the absorbance/reflection mode at 550 nm using standard mixtures of PC, PA and PG. The aqueous phase did not contain detectable amounts of phospholipids. Activity of the enzymes was determined

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from the increase of PG or PA, respectively, within the linear range of the progress curve. 2.8. SDS-PAGE For determination of the apparent molecular masses of the purified proteins, electrophoresis in the presence of SDS was carried out on a Bio-Rad Mini Protean II dual vertical slab gel electrophoresis cell (Bio-Rad Laboratories) according to Ref. [17] by using 7.5% polyacrylamide gels. Proteins were stained with silver according to Ref. [18]. 2.9. Determination of isoeletric point To determine the isoelectric point of PLD-A and PLD-B, IEF was carried out in a PhastGel IEF 3-9 on PhastSystem Separation and Control Unit (Pharmacia LKB Biotechnology). For calibration of pI IEF-markers liquid mix 3 – 10 was used. Proteins were stained with Coomassie brilliant blue G-250. 2.10. Glycoprotein detection A 7.5% SDS-PAGE gel containing PLD-A, PLD-B, peroxidase (as standard of a glycosylated protein) and aldolase (as standard of a non-glycosylated protein) was blotted onto a nitrocellulose blotting membrane at 300 V and 5 mA/cm2 for 180 min in a Fast-Blot apparatus (Biometra). After protein transfer (checked by staining with Ponceau S) the proteins were assayed for total carbohydrate by using the ECL glycoprotein detection module. Colour detection of carbohydrates was performed with 0.06 M Tris containing 3,3V-diaminobenzidine (0.7 mg/ml) and urea hydrogen peroxide (0.2 mg/ml). The deglycoslation of glycoproteins was performed with N-glycosidase F according to the ECL glycoprotein detection module protocol.

Fig. 1. Elution profile of PLD from poppy seedlings by Ca2 +-mediated hydrophobic interaction chromatography. PLD containing solution was applied onto Octyl-Sepharose CL-4B column in the presence of 50 mM CaCl2. Elution was performed by using of (I) 0.01 M sodium acetate buffer/50 mM CaCl2 (pH 5.5), (II) 0.005 M sodium acetate buffer/30 mM CaCl2 (pH 5.5) and (III) 0.005 M sodium acetate buffer/0.1 mM EDTA (pH 5.5) as described in Section 2.4. The PLD activity was determined toward PpNP as described in Section 2.6. (—) absorbance at 280 nm; (- - -) elution buffers I, II, III; (- -.- -) PLD activity at pH 8.0; ( – D – ) PLD activity at pH 5.5.

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2.11. N-terminal amino acid sequencing

Table 1 Purification of PLD from poppy seedlings

N-terminal amino acid sequencing was performed with a 492 cLC Protein Sequencer (PE Applied Biosystems).

Purification step

3. Results 3.1. Identification of PLD in poppy and purification of two enzyme forms Examining poppy seedlings for PLD activity, we found that this enzyme is formed during the first days of germination and is located in endosperms as well as in the developing seedlings. The activity toward the synthetic substrate PpNP was highest in 2-day-old endosperms that were used for purification. A fast isolation and purification of PLD was possible by the Ca2 +-mediated hydrophobic interaction chromatography as developed for the purification of PLD from cabbage [19]. This chromatographic step was performed after an ammonium sulfate precipitation with subsequent dialysis. In the presence of CaCl2, PLD was bound to the hydrophobic gel. By removing Ca2 + ions with 0.1 mM EDTA two enzyme forms were eluted, which were active at pH 8.0 (PLD-A) or at pH 5.5 (PLD-B). Fig. 1 shows the elution profiles. In accordance with the activation of PLD by Zn2 + ions as reported below, CaCl2 in the buffer solutions could be replaced by ZnCl2 with the same purification results. Both enzymes were homogeneous in SDS-PAGE (Fig. 2).

Fig. 2. SDS-PAGE of PLD isolated from poppy seedlings. Lane 1: crude extract after homogenization of the acetone powder and centrifugation at 12,000  g; lane 2: enzyme solution before Ca2 +-mediated hydrophobic interaction chromatography; lane 3: unified fractions no. 33 – 36 after Ca2 +mediated hydrophobic interaction chromatography (PLD-A); lane 4: unified fractions no. 39 – 42 after Ca2 +-mediated hydrophobic interaction chromatography (PLD-B); lane 5: HMW protein standards (53 – 220 kDa as indicated in the figure).

Crude extracta (NH4)2SO4 precipitateb

Protein [mg]

43.25 9.56

Octyl-sepharose CL-4B Fractions 33 – 36 0.1432 (PLD-A) Fractions 39 – 42 0.1827 (PLD-B)

Activity [Amol min

1

]

Specific activity [Amol min 1 mg

1

pH 8.0

pH 5.5

pH 8.0

3.7369 1.9897

4.6538 2.3847

0.0864 0.2081

0.1076 0.2494

1.0482

–

7.3198

–

–

1.8507

–

]

pH 5.5

10.1297

The hydrolytic PLD activity was determined toward PpNP at pH 8.0 and at pH 5.5 as described in Section 2.6. a After homogenization of the acetone powder and centrifugation at 12,000  g as described in Section 2.4. b After centrifugation of precipitate at 26,000  g and subsequent dialysis as described in Section 2.4.

Table 1 summarizes the data of purification. The purification factors for the two isoenzymes were 84.7 (PLD-A) and 94.1 (PLD-B). 3.2. Protein characterization of the two PLD forms From SDS-PAGE, the molecular masses of PLD-A and PLD-B were estimated to be 116.4 and 114.1 kDa. The isoelectric points were 8.7 (PLD-A) and 6.7 (PLD-B). PLDA and PLD-B were shown to be glycosylated (Fig. 3). A positive deglycosylation by using N-glycosidase F indicated the existence of N-linked carbohydrate in both cases. Nterminal protein sequencing of the two enzymes was not possible, indicating an N-terminal blockage.

Fig. 3. Detection of carbohydrate moiety by using ECL glycoprotein detection module. Membrane after total carbohydrate detection stained as described in Section 2.10. Lane 1: PLD-A; lane 2: PLD-B; lane 3: peroxidase as a glycosylated reference, lane 4: aldolase as a non-glycosylated reference.

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Fig. 4. Activity of PLD-A and PLD-B as a function of pH value. The hydrolytic activity of PLD-A (.) and PLD-B (D) was determined at different pH values as described in Section 2.6. Values are means F S.D. (n = 3).

3.3. pH-activity profiles of PLD-A and PLD-B Fig. 4 demonstrates the strong differences in the hydrolytic activities of PLD-A and PLD-B toward PpNP as a function of pH value. PLD-A has a distinct pH optimum at pH 8.0, where the activity of PLD-B is insignificant. On the other hand, PLD-B is most active at pH 5.5, where PLD-A is nearly inactive. The maximum activity of PLD-B is higher than that of PLD-A by 38% under the conditions of the assay. 3.4. Activation of PLD-A and PLD-B by metal ions Since most plant PLDs described so far are activated by Ca2 + ions [20], purified enzymes were assayed as a function of the concentration of CaCl2. PLD-A as well as PLD-B were inactive in the absence of added Ca2 + ions and showed maximum activity at 10 mM CaCl2 (Fig. 5). The activation by metal ions was also studied with Zn2 + and Mg2 + ions. While the activation of the two enzymes by Mg2 + ions was

marginal, Zn2 + ions proved to be more activating than Ca2 + ions for PLD-B (Fig. 5). At the optimum Zn2 + concentration (5 mM), PLD-B was four times more active than at the optimum Ca2 + concentration. The activity of PLD-A could not be determined in the presence of Zn2 + ions because of the precipitation of zinc hydroxide at pH 8.0. 3.5. Transphosphatidylation and hydrolytic activities of PLD-A and PLD-B in two-phase systems The transphosphatidylation potentials of PLD-A and PLD-B were compared in a biphasic system consisting of sodium acetate buffer (pH 5.5) or Tris – HCl buffer (pH 8.0) containing 40 mM CaCl2, diethyl ether with PC as substrate and glycerol as acceptor alcohol. The reaction was followed by HPTLC with densitometric quantification of the products. As demonstrated in Fig. 6, PLD-B has a high transphosphatidylation potential at pH 5.5. More than 80% of PC were converted into the transphosphatidylation product PG after 240 min, whereas no PA was detected under these

Fig. 5. Activity of PLD-A and PLD-B as a function of Ca2 + and Zn2 + ion concentration. The hydrolytic activity of PLD-B was determined at pH 5.5 in the presence of CaCl2 (o) and ZnCl2 (.) as described in Section 2.6. The hydrolytic activity of PLD-A was determined at pH 8.0 in the presence of CaCl2 (D) as described in Section 2.6. Values are means F S.D. (n = 3).

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Fig. 6. Transphosphatidylation of PC by purified PLD-B in two-phase systems. The conversion of PC (.) into PG (D) was determined by HPTLC as described in Section 2.7. Values are means F S.D. (n = 3).

reaction conditions. The initial rate of transphosphatidylation was 4.86 F 0.50 Amol min 1 mg 1. In the absence of glycerol, hydrolysis proceeded with an initial rate of 0.342 F 0.051 Amol min 1 mg 1. At pH 8.0 neither transphosphatidylation nor hydrolysis was detected with PLD-B. In contrast, PLD-A showed no transphosphatidylation activity at pH 5.5 or pH 8.0. In the absence of glycerol the hydrolytic activity was observed at pH 8.0 with an initial rate of 0.167 F 0.036 Amol min 1 mg 1.

4. Discussion Although poppy seedlings are an interesting model object both for the study of lipid metabolism and secondary metabolite production (opium alkaloids) [13]; there have been no references about PLD activity in poppy before this study. Interestingly, the purification of PLD revealed the existence of two enzyme forms. Both enzymes (PLD-A and PLD-B) could be purified to homogeneity in two steps only by Ca2 +-mediated hydrophobic interaction chromatography after ammonium sulfate precipitation (Figs. 1 and 2, Table 1). This method was successfully applied for the purification of several plant PLDs before [19,21,22]. Interestingly, Zn2 + ions fulfilled the same function as Ca2 + ions in the purification procedure, which is in correspondence with the surprising activation of PLD-B (Fig. 5) and suggests that the effects of metal ions on enzyme activity and carrier binding are closely related. The finding of two forms of PLD is in accordance with the occurrence of multiple forms of PLD in other plants such as castor bean [23], Arabidopsis [2], cabbage [24] and tomato [25]. However, PLD-A and PLD-B strongly differ from each other as well as from other plant PLDs. From the different pH optima (pH 8.0 for PLD-A and pH 5.5 for

PLD-B) and the great differences in their transphosphatidylation potential, it can be concluded that PLD-A and PLD-B are isoenzymes originating from different genes. Both enzymes differ in their isoelectric points (8.7 for PLD-A and 6.7 for PLD-B) by two pI units. The experimentally determined molecular masses of the two enzymes are similar (114.1 and 116.4 kDa), but distinctly higher than those of most plant PLDs, which are between 90 and 95 kDa [26,27]. On the other hand, two PLD genes of the most common a-type have been identified in poppy seedling on the basis of cloning and sequencing cDNA fragments prepared from mRNA [28]. This discrepancy might be explained by the detection of a carbohydrate content of PLD-A and PLD-B (Fig. 3), which has been not described for other plant PLDs hitherto. For PLD from rapeseed a molecular mass of 105 kDa was reported [29]. Evident differences from other plant PLDs are given with respect to the metal ion activation (Fig. 4). Thus, Zn2 + ions are better activators of PLD-B than Ca2 + ions, which is unusual for plant PLDs [1]. Also the optimum Ca2 + concentration of poppy PLD-A and PLD-B (10 mM) differs from that of other plant PLDs. It is lower than for the common a-type of PLD (20 – 100 mM) and higher than for the h- or g-type of PLDs (about 50 AM) [2]. The most striking finding is the great difference in the catalytic properties of PLD-A and PLD-B. The high transesterification potential of PLD-B, on one hand, and the inability of PLD-A to catalyze the transphosphatidylation as well as the pH-optimum of PLD-A at pH 8.0, on the other hand, are unusual for plant PLDs. Therefore, poppy PLDs might provide key information on the biological function of PLDs, particularly the function of the transphosphatidylation activity. Clarification of differences between the two isoenzymes will possibly provide indications for their different physiological role in poppy seedlings. Moreover, PLD-B

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should be attractive for biotechnological application in phospholipid transformation.

[14] [15]

Acknowledgements The authors wish to thank Dr. Nadeshda Dittrich for her help in the HPTLC experiments and Dr. Karl-Peter Ru¨cknagel for N-terminal amino acid sequencing. The financial support by Slovak Grant Agency VEGA 1/ 8211/01 and by Grant FaF UK/1671/2000, Bratislava, Slovakia as well as by the German Academic Exchange Service (DAAD), Bonn, Germany is gratefully acknowledged.

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