Detection of Glycolipids as Biotinylated Derivatives Using Enhanced Chemiluminescence

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

241, 59–66 (1996)

0378

Detection of Glycolipids as Biotinylated Derivatives Using Enhanced Chemiluminescence Luis E. Herna´ndez,1 Nicholas J. Brewin, and Bjørn K. Drøbak John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom

Received February 26, 1996

A new method for the detection of glycolipids as biotinylated derivatives is presented. This method is based on the detection of lipids by enhanced chemiluminescence (ECL) following conjugation with streptavidin–horseradish peroxidase (SHRP). Partial biotinylation of glycolipids is achieved after mild oxidation of the glycan moiety with Na–meta-periodate to increase the availability of biotin-reactive sites. There are several significant advantages of the glycolipid ECL detection: it avoids the need for radiolabeling; it provides the possibility of antibody probing; it allows reprobing of SHRP conjugate to adjust background and luminol light emission levels; it permits easy and accurate quantification of glycolipids at low concentrations; and it involves nondestructive staining, thereby enabling further molecular analysis. q 1996 Academic Press, Inc.

Glycolipids are known to be involved in a very wide range of cellular events. Two major families are the glycosphingolipids (1) and glycophosphatidylinositol protein anchors (2). Involvement of glycolipids in diverse biological processes, such as gastrulation in vertebrate embryos (3), adherence of cell surfaces (4, 5), and stabilization of membrane structures (6) have been described. Due to the complexity of glycolipid structure, many functional aspects remain to be elucidated. Comparatively few methods are available for the general detection of glycolipids. Most of these methods are destructive (involving use of dye–reagents in concentrated H2SO4 ), have low sensitivity, or depend on radiolabeling. Immunodetection is a sensitive method to detect certain glycolipids, but it depends on the availability of a specific antibody which recognizes a 1 To whom correspondence should be addressed. Fax: 01603456844.

particular antigen. The blotting of glycolipids onto PVDF2 membranes overcomes the problems of silica gel contamination of lipid samples and provides increased sensitivity, even when established techniques of detection are employed (7, 8). An additional advantage of blotting glycolipids onto PVDF membranes is that it becomes much easier to detect them by antibody recognition without the problems encountered using plasticcoated TLC plates for affinity analysis (9). Many different molecules of biological interest, such as nucleic acids, antibodies, and lectins, have been derivatized with biotinylating reagents (10). Currently there are many applications in which biotinylated biomolecules are detected by conjugation with streptavidin – horseradish peroxidase. In particular, peroxidation of luminol and recording of its light emission has been employed as a method of chemiluminescence detection (11). Here we report the development of a method for glycolipid detection based on partial biotinylation of mildly oxidized carbohydrate – lipid moieties. Periodate-pretreated lipids are first derivatized with a biotinylating reagent and then resolved by high-performance thin-layer chromatography (hptlc), blotted onto PVDF membranes, and detected as biotinylated glycan derivatives using streptavidin – horseradish peroxidase and enhanced chemiluminescence (ECL). MATERIALS AND METHODS

Lipid Standards Authentic lipid standards were prepared by dissolving 1 mg lipid per milliliter of methanol:chloroform 2

Abbreviations used: DeCCE, deacylated carrot cell extract; ECL, enhanced chemiluminescence; HPLC, lipid fraction collected from the HPLC; JIM, John Innes monoclonal antibody; PVDF, polyvinyldene difluoride; SHRP, streptavidin–horseradish peroxidase; BSA bovine serum albumin; PBS, phosphate-buffered saline; TBS, Trisbuffered saline.

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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(1:1, v/v). A range of commercially available lipids was used (Sigma Co., St. Louis, MO): L-a-phosphatidylethanolamine (PE, di 18:0); L-a-phosphatidylinositol (PI, 18:2 and 16:0); DL-a-phosphatidyl-L-serine (PS, di 18:0); L-a-phosphatidylcholine (PC, lecithin from soybean enriched in 16:0, 18:1, and 18:2); sphingomyelin (Sph, 18:0); and mixtures of galactosylcerebrosides (GalCer), glucosylcerebrosides (GlcCer), and lactosylcerebrosides (LacCer). A commercially available glycolipid standard mixture (Calbiochem, Code No. 361905, UK) was used for ECL detection experiments involving glycolipids with more than four monosaccharide units. The mixture contained galactosylcerebroside (Galb1–1Cer), lactylceramide(Galb 1–4Glc1–1Cer), trihexosylcera mide (Gala1–4Galb1–4Glcb1–1Cer), globoside (GalNAcb1–3Gala1–4Galb1–4Glcb1-1Cer), and Forssman glycolipid (GalNAca1–3GalNAcb1–3Gala1–4Galb1– 4Glcb1–1Cer). It was prepared by dissolving 1 mg per milliliter in methanol:chloroform (1:1, v/v). Lipid Oxidation and Biotinylation The flow chart of the procedure is shown in Fig. 1. Lipid extract (25 ml, in methanol:chloroform, 1:1, v/v) was transferred to a soda-glass vial. Reaction buffer was added (225 ml of 100 mM Na acetate/acetic acid buffer, pH 5.5) and the mixture sonicated for a few seconds, allowing a suspension to form. Sodium metaperiodate was added (150 ml of 0.5 mg ml01 in reaction buffer) and the vial was vortexed thoroughly. Vials were covered with aluminium foil and the mild-oxidation reaction was allowed to proceed for 15 min at room temperature. The meta-periodate reacts with the carbohydrate portion to form aldehyde groups that react spontaneously with biotin hydrazide. The reaction was stopped by adding 150 ml sodium bisulfite (20 mg ml01 in reaction buffer) and samples were incubated for 10 min at room temperature to eliminate excess meta-periodate. Biotinylation was initiated by adding 25 ml of 5 mM biotin hydrazide (Sigma Co.) in dimethylformamide to the sample mixture, and the vials were incubated for 3 h at room temperature. Lipids were recovered after sequential addition of 1 ml of methanol:chloroform (1:1, v/v) and 500 ml chloroform followed by recovery of the organic phase. The solvent was then evaporated under O2-free nitrogen. Thin-Layer Chromatography, Transfer to PVDF Membrane, and Treatment of Lipids with Streptavidin Conjugate The dried biotinylated glycolipids were redissolved in 50 ml chloroform:methanol (1:1, v/v) and loaded onto a hptlc plate (60 A HPK Whatman, UK), preimpregnated with 1% potassium oxalate, 1.5 mM EDTA

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and activated for 2 h at 1207C before use. The plate was developed using the following solvent system: chloroform:methanol:ammonia:water (45:35:2:8, v/v/ v/v). The hptlc plate of the commercial standard mixture of glycolipids was developed using a solvent system consisting of chloroform:methanol:H2O (65:25:4, v/v/v), according to the manufacturer’s instructions (Calbiochem) to identify the bands corresponding to the individual glycolipid species present. After air drying, hptlc-separated samples were blotted onto a PVDF membrane (Hybond – PVDF, Amersham, UK) essentially as described by Taki et al. (7, 8). In brief, after soaking the silica plate in blotting solvent (propan-2-ol:methanol:0.2% aqueous CaCl2 , 40:20:7, v/v/ v) for 15 s, lipids were transferred onto the blot by pressing the plate and membrane with a hot laundry iron (80 – 1007C) for 1 min. The PVDF membrane was separated from contact with the iron by the presence of a sheet of glass-fiber (GF/A Whatman, UK). Successful transfer of samples onto the membrane was monitored by spraying it with primuline fluorescent reagent (0.5% w/v in water) followed by visualization under uv light (7). Before treatment with streptavidin–horseradish peroxidase (SHRP), the blot was moistened with methanol:water (75:25 v/v) and then blocked in 3% BSA (w/v) in phosphate-buffered saline (PBS, 100 mM Na2HPO4/NaH2PO4 and 100 mM NaCl, pH 7.5) for 2 h at room temperature or overnight at 47C. Following thorough rinsing of blocking solution with PBS (at least 6 rinses of 75 ml for 30 min), the membrane was incubated for 3 h with SHRP (Amersham) diluted 1/2000 in PBS. Before ECL detection, the membrane was again thoroughly washed with PBS as described above. ECL reagent (Amersham) was prepared from solutions A and B, mixed 1:1; according to the manufacturer’s instructions. Before use, it was usually diluted fivefold in deionized water and poured onto the PVDF membrane and the blot was exposed to radiograph film (Fuji RX, Japan).

Quantification of Glycolipid Concentration Following the procedures outlined in Fig. 1, ECL films were scanned in a HP ScanJet 3C/ADF using the DeskScan II HP software. Optical density of peak areas (given as scanner units, i.e., number of pixels) was quantified by the ImagQuant 3.3 software. Results are the mean of 15 replicates from 5 independently blotted GalCer standards (0.5, 1.0, and 5.0 mg, 3 replicates per blot). Each blot was incubated for 1 min with the ECL reagent (solution A:solution B:H2O, 1:1:10, v/v/v) and the film was exposed for 1.5 min.

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FIG. 1. Flow chart of the glycolipid biotinylation procedure, hptlc separation and PVDF blotting. For further details, see Materials and Methods.

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Staining of Lipids and Glycolipids in hptlc Plates Nonspecific staining of lipids was carried out by spraying the hptlc plate with 3% (w/v) cupric acetate in 8% H3PO4 (v/v) and heating at 1807C for 1 h. Glycolipids were visualized by fine-spraying with orcinol reagent (0.2 g liter01 orcinol in 75% H2SO4 ) and heating at 1207C for 15 min, according to Ref. (12). Cell Culture, Lipid Extraction Carrot (Daucus carota L.) cv. ‘‘Oxford’’ suspension culture cells were maintained as described by McCann et al. (13). Whole-cell acidic lipid extracts were prepared by homogenization of cells in 15 ml of quench solvent (chloroform:methanol:concentrated HCl, 10:10: 0.07, v/v/v). The aqueous phase was removed and the lower-organic phase was back-washed with 4 ml aliquots of washing solvent (methanol:0.6 M HCl:chloroform, 9.6:9.4:0.6, v/v/v). After centrifugation, the organic phase was recovered and the solvent was evaporated under nitrogen. This extract was subjected to mild-alkaline deacylation with 4 ml monomethylamine, prepared as described by Drøbak and Roberts (14), in order to make possible the separation of glycoglycerophospholipids (sensitive to deacylation) from glycosphingolipids (insensitive to deacylation) by aqueous/n-butanol-solvent partitioning (14). The lipids in the organic phase (described as deacylated carrot cell extract, DeCCE) were resolved by adsorption high-performance liquid chromatography (hplc; Adsorbsphere 3 mm, 150 1 4.6 mm, Alltech) using a binary gradient system (solvent A, n-hexane:propan-2-ol:H2O, 578:390:32 v/v/v; and solvent B, n-hexane:propan-2-ol:H2O, 470:470:60, v/v/v) at 0.9 ml min01 flow rate. Fractions (0.45 ml) were collected from 20 to 27.5 min. Further details are described in Herna´ndez et al. (15). Immunodetection with JIM 18 The same blot used for glycolipid ECL detection was probed with the JIM 18 (rat IgG monoclonal antibody), which recognizes a widespread plant glycolipid antigen (16). The blot was rinsed in Tris (tris[hydroxymethyl]aminomethane)-buffered saline (TBS, 100 mM Tris/HCl and 100 mM NaCl, pH 7.6) and blocked with 3% BSA (w/v) in TBS for 3 h at room temperature. The membrane was subsequently incubated overnight at 47C with JIM 18 antibody (as hybridoma cell culture supernatant, diluted 1/100 with 3% BSA w/v in TBS). The membrane was thoroughly rinsed with at least 3 vol of TBS (75 ml for 30 min) and incubated with a secondary antibody (IgG goat anti-rat, Sigma) conjugated with alkaline phosphatase (diluted 1/2000 with 3% BSA w/v in TBS) for 3 h at room temperature. The membrane was thoroughly washed with TBS prior to stain-

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ing with 20 ml stain solution (0.1 mg ml01 nitroblue tetrazolium, 0.02% dimethyl sulfoxide, 0.05 mg ml01 5bromo-4-chloroindolyl phosphate, 4 mM MgCl2 in 100 mM Tris/HCl, pH 9.6). RESULTS AND DISCUSSION

Three commercial glycolipids were used as standards for the ECL–glycolipid detection method which is outlined in Fig. 1. Each of these lipid samples contains a mixture of components, which explains the multiband pattern observed in the radiograph film (Fig. 2A). Biotinylation and SHRP conjugation detected glycolipids at the concentration used (5 mg ml01 ), whereas other lipids (sphingolipids and glycerolipids containing saturated and nonsaturated fatty acid acyl chains) were not visualized. Cu–acetate/H3PO4 staining of lipids showed that the mobility of these glycolipid bands was unaffected by mild oxidation or biotinylation (Fig. 2B). Similar results were obtained with mildly oxidized biotinylated 33P-labeled crude carrot cell extracts, where there were no changes in mobility observed for metabolically radiolabeled lipids (data not shown). GalCer, GlcCer, and LacCer were also visualized using the orcinol/H2SO4 reagent, showing them as purple bands (Fig. 2C). The mobility was again found to be unaffected by the treatments and the ECL-detection procedure, compared with their respective controls (Fig. 2D). Glycolipids could often be stained by SHRP following treatment with biotin hydrazide without pretreatment of the samples with meta-periodate (lane 10, Fig. 2A). A low level of staining was observed, which probably reflects the presence of some free aldehyde groups in the lipid samples. Nevertheless, the intensity of the bands was significantly enhanced by mild oxidation with Na–periodate (lane 9, Fig. 2A). Lipids that were neither mildly oxidized nor biotinylated did not give rise to ECL emission (lane 11, Fig. 2A), indicating a specific binding of SHRP with biotinylated glycolipid derivatives. The quality of bands may be somewhat affected by the blotting procedure. During heating the solvent used for the lipid transfer to the PVDF membrane boils (temperature above 1007C). There is little one can do to overcome this problem but it clearly does not affect the validity of the method per se. This can be observed in Refs. (7, 8, 17). Previously, biotinylation experiments were performed after blotting of glycolipids to the PVDF membrane. The oxidation with meta-periodate and biotinylation were then carried out on the membrane. In this case, we found that the sensitivity was much less and there was a high background, probably caused by the reaction of the membrane itself (data not shown). Therefore, in subsequent experiments the lipids were

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FIG. 2. Comparison of the glycolipid ECL-detection procedure with currently used alternative methods. Lipids were biotinylated and then resolved by hptlc. (A) ECL detection of glycolipids, 5 mg of each lipid; (B) Cu–acetate/H3PO4 detection of lipids, 5 mg of each lipid; and (C) orcinol/H2SO4 detection of glycolipids, 25 mg of each lipid: 1, GalCer; 2, GlcCer; 3, LacCer; 4, Sph; 5, PI; 6, PC; 7, PE; 8, PS; 9, mixture of standards with complete treatment; 10, mixture without mild-oxidation treatment but biotinylated; 11, mixture without mild-oxidation and biotinylation treatments. (D) Orcinol/H2SO4 detection of GalCer, GlcCer, and LacCer, 25 mg of each, with: (a) mild oxidation and biotinylation, (b) only biotinylation, and (c) without these treatments.

biotinylated before being transferred to the PVDF membrane. A commercial mixture of glycolipids, which included lipids with more than four saccharide residues, was used to investigate the general applicability of the biotinylation procedure. As shown in Fig. 3A, ECL detection was better using lipids biotinylated after mild meta-periodate treatment (lane 1). Lipids with larger saccharide moieties (ú3 monosaccharides) were biotinylated without mild oxidation of hydroxyl groups (lane 2) but, as expected, no signal was recorded from lipids not biotinylated (lane 3). It is possible that the large glycolipids contain biotinreactive aldehyde groups, and therefore no prior mild oxidation is necessary. These results are in agreement with those of Fig. 2. Nonspecific staining with Cu – acetate/H3PO4 (Fig. 3B) indicates that the mobil-

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ity of the standard lipids was not altered by the biotinylation procedure (lanes 1 – 3), confirming previous results (Fig. 2). Furthermore, the partitioning of all glycolipids into the organic phase during the biotinylation procedure was satisfactory, when compared to a control sample of lipids (starting material) that was directly loaded on the hptlc (lane 4). It is possible that carbohydrate moieties of glycolipids would be derivatized in such a way that no vicinal hydroxyl groups are available as reactive sites for biotin conjugation after meta-periodate treatment. However, in most known glycolipids it is feasible to find at least one such reactive site, as observed with the highly derivatized large glycolipids tested (Fig. 3). The ECL–glycolipid detection procedure was compared with that of the orcinol/H2SO4 detection system. Figure 4 shows that the sensitivity of the ECL method

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FIG. 3. Detection of a glycolipid standard mixture (5 mg per lane) using (A) ECL and (B) Cu–acetate staining: (a) Galb1–1Cer, (b) Galb1–4Glcb1–1Cer, (c) Gala1–4Galb1–4Glcb1–1Cer, (d) GalNAcb1–3Gala1–4Galb1–4Glcb1-1Cer, and (e) GalNAca1–3GalNAcb1–3Gala1–4Galb1–4Glcb1–1Cer. Lanes: (1) mildly oxidized and biotinylated, (2) biotinylated, (3) neither treatment, and (4) starting material, lipids without any treatment at all directly loaded onto the hptlc plate.

was enhanced approximately 20-fold relative to the orcinol/H2SO4 procedure (Table 1). That the sensitivity can be further increased by altering the composition of the ECL reagent and time of exposure is shown in Fig. 4B and 4C. This is an important advantage because it opens the possibility of reprobing the blot several times in order to optimize the sensitivity. Furthermore, the membrane can be stored for further analysis. For example, after removal of the ECL reagent by rinsing with deionized water, the membrane can be kept active for at least 1 week at 47C in PBS (data not shown). Moreover, the concentrated H2SO4 used in the orcinol reagent leads to rapid loss of the glycolipid staining from the hptlc plate or the membrane (7), whereas the results of the ECL procedure are preserved as a permanent and high-contrast record on film. Varying quantities of GalCer, used as a glycolipid standard, were subjected to mild oxidation, biotinylation, and ECL detection. By using an image scanner, the intensity of spots (measured as peak areas) indicated that the ECL method for glycolipid detection is quantitative. The standard curve obtained was reproducible (g2 Å 0.99) at the standard concentrations used (Fig. 5). Above 5 mg GalCer, saturation was reached in the luminol–light emission profile (data not shown). Nevertheless, it is possible to reduce the signal emission by diluting the ECL reagent with deionized H2O, and reusing the same blot until an optimum signal is obtained. As well as detecting glycolipids in a mixture of standard lipids, we have also used the ECL method described in this paper to monitor the purification of

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plasma membrane glycolipids from lipid extracts of plant tissues. Thus, a deacylated carrot cell extract was fractionated by hplc (15). DeCCE and hplc-fractionated samples (HPLC) were subjected to biotinylation and resolved by hptlc (Fig. 6A, lanes a), following the procedure described in Fig. 1. As a control, DeCCE and HPLC samples were also resolved in parallel, but without being subjected to the biotinylation step (lanes b). Following detection by SHRP and ECL it was found that the nonbiotinylated samples contained material that reacted with the streptavidin probe. There was a dense band with high mobility (close to the solvent front) in the DeCCE which was also recognized in the nonbiotinylated sample (empty arrow, Fig. 6A). This dense band did not correspond to material that could be detected by the orcinol/H2SO4 staining of glycolipids (as purple bands, data not shown), indicating that it probably did not contain glycolipids. Apparently, unknown components from the deacylated plant lipid extract (possibly carotenoids, sterols, or alkaloids) may mimic the biotin structure and react directly with SHRP. These streptavidin-reactive bands represented major components of the crude carrot cell lipid extract after deacylation, but were much less intense in the HPLC fraction, indicating that hplc could be used to eliminate these cross-reacting components. In any case, these compounds were resolved from the putative glycolipids in both fractions by the hptlc purification step (Figs. 6A and 6C). Therefore, to ensure that biotinylated derivatives conjugate specifically with SHRP, appropriate controls should be used (i.e., with and without biotinylation treatment), in particular when plant lipid extracts are investigated. To reduce nonspecific binding, a preliminary purification step could be included. Thus, steroids and/or carotenoids could be

FIG. 4. Comparison of sensitivity using orcinol/H2SO4 (A) and ECL (B, C) glycolipid-detection methods with decreasing concentrations of GalCer. (B) The blot was treated with ECL reagent (solvent A:solvent B:H2O, 1:1:10, v/v/v) and exposed for 1.5 min. (C) The same blot was reprobed with ECL reagent (same components, 1:1:5, v/v/v) and exposed for 1 min.

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DETECTION OF GLYCOLIPIDS BY ENHANCED CHEMILUMINESCENCE TABLE 1

Comparison of Glycolipid–Orcinol/H2SO4 Staining and Glycolipid–ECL Detection Intensity of bands from left to right (pixels)

Orcinol (A) ECL (B) ECL (C)

1

2

3

4

5

6

7

8

548 1078 9576

425 630 3666

318 530 2082

147 520 1970

99 325 1392

38 207 1050

— 50 922

— — 31

Note. Data show the intensity of bands following treatments A, B, and C, as described in the legend to Fig. 4, using scanner image quantification.

easily separated from glycolipids due to their lower polarity (stippled arrowhead, Fig. 6C). One of the advantages of the lipid detection method described here is its compatibility with standard systems for immunodetection of glycolipids. Glycosphingolipids elicit a highly antigenic response, and many antibodies have been obtained from glycolipids extracted from different organisms (3, 18, 19). For example, in our laboratory (16) we have obtained a monoclonal antibody (JIM 18) which recognizes a putative inositol-containing glycosphingolipid that we are currently purifying. Thus, in conjunction with ECL detec-

FIG. 5. Quantification of glycolipids by the ECL system. (A) Radiograph of three replicates of GalCer (concentrations: 0.5, 1.0, and 5.0 mg) and detected with ECL (1:1:10, reagent A, B, and H2O exposed for 1.5 min). (B) Standard curve of mean optical density versus GalCer concentrations. Bar indicates standard error of 15 replicates (g2 Å 0.99, P õ 0.05).

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tion of glycolipids, fractions were analyzed with JIM 18 antibody. The results shown in Fig. 6 indicate that a relatively pure lipid fraction was obtained after separation of samples by hplc, as most other lipid bands disappear (Fig. 6C) in the region where JIM 18 antigen is localized (double arrowheads, Fig. 6B). ECL detection showed a smear in the HPLC fraction corresponding to the region where JIM 18 recognized two compounds. Further analysis of both bands by secondary ion mass spectrometry revealed that there was a single component (single peak, data not shown). In addition, the glycolipid band present in DeCCE (single arrowhead, Fig. 6A) with greater mobility than JIM 18, was not present in the HPLC fraction. It is known that meta-periodate modifies carbohydrate epitopes by forming alditols which react with biotin hydrazide, but conceivably this process could also damage antigenicity. However, the concentration of meta-periodate used for glycolipid biotinylation is lower than the one normally used for screening of carbohydrate antigens with antibodies. Nevertheless, it was observed that, when the aqueous solubility of glycolipids in the reaction buffer was enhanced by adding increasing concentrations of methanol, the antibody– antigen recognition was gradually lost (data not shown). A similar effect was obtained when the concentration of meta-periodate was increased 100-fold. In conclusion, there are several significant advantages of this new method for glycolipid detection: (1) it avoids the need for radiolabeling; (2) it has a much wider and more general applicability than immunodetection because it only requires a free aldehyde group or a meta-periodate-sensitive site; (3) it provides the additional possibility of antibody (or lectin) probing; (4) it allows reprobing of SHRP conjugate to adjust background and luminol light emission levels; (5) it permits easy and accurate quantification of glycolipids at low concentrations; and (6) it involves nondestructive staining, thereby enabling further molecular analysis (e.g., by mass spectrometry). We are currently investigating the possibility that direct mass-spectrometric analysis can be carried out after ECL detection.

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FIG. 6. Detection of glycolipids from plant lipid extracts. (A) ECL system, (B) recognition with JIM 18 antibody, and (C) nonspecific staining using Cu–acetate. Monomethylamine-deacylated carrot cell extract (DeCCE) and a hplc-purified fraction (HPLC). (a) Mildly oxidized and biotinylated lipids and (b) untreated lipid samples. Stippled arrowhead, unspecific conjugation with SHRP; single arrowhead, major glycolipid component of DeCCE; double arrowhead, band detected by ECL procedure and JIM 18 immunostaining.

ACKNOWLEDGMENTS We thank Dr. R. Gallagher (Department of Chemistry, University of Warwick, UK) for help with secondary ion mass spectrometry and Dr. S. Perotto (Department of Plant Biology, University of Torino, Italy) for general discussion. We are indebted to Amersham UK Ltd. for supplying reagents and probes used in this work. L.E.H. was supported by EC HCM Grant CHRX CT940699.

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