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Journal of Integrative Plant Biology Formerly Acta Botanica Sinica 2005, 47 (1): 50−59

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Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit Qiu-Hong PAN, Ke-Qin ZOU, Chang-Cao PENG, Xiu-Ling WANG and Da-Peng ZHANG* (State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China)

Abstract: The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1. 26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively. These results provide a substantial basis for the further studies of the acid invertases in apple fruit. Key words: antiserum; cell wall-bound acid invertase; fruit; Malus domestica; purification; soluble acid invertase.

Sucrose is the predominant export photoassimilate from leaves and plays an important role in carbohydrate transportation and metabolism in higher plants (Quick and Schaffer 1996; Patrick 1997). Acid invertase (β-fructosidase; EC 3.2.1.26), an enzyme catalysing the irreversible cleavage of sucrose into glucose and fructose, is one of the key enzymes in sucrose metabolism in apple fruit (Beruter 1985; Beruter et al. 1997), as in sink organs of other plants (Quick and Schaffer 1996). We recently reported that photoassimilate unloading from the phloem in apple fruit follows an apoplasmic pathway (Zhang et al. 2001, 2004; Peng et al. 2003), where acid invertases play a key role in regulating sucrose unloading in the fruit. Acid invertases can be categorized into two types in terms of their different cellular compartmentation, one being located in the apoplastic space, called cell wallbound acid invertase (CWI), and the other being

located in the vacuole, called soluble acid invertase (SAI; Quick and Schaffer 1996; Patrick 1997). Many studies have been devoted to the purification and determination of the biological functions of acid invertases in other plants (Karuppiah et al. 1989; Fahrendorf and Beck 1990; Iwatsubo et al. 1992; Klann et al. 1996; Isla et al. 1999; Tang et al. 1999). However, acid invertase has not been purified to date in apple fruit. In the present study, we report on the purification of two forms of acid invertase, namely CWI and vacuole SAI, and the characterization of their biochemical and immunological properties.

1

Materials and Methods

1.1 Plant materials Apple (Malus domestica Brohk. cv. Starkrimson) fruits were harvested from 8- to 9-year-old trees. The fruits were processed immediately or frozen in liquid

Received 23 Jun. 2004 Accepted 24 Oct. 2004 Supported by the National Natural Science Foundation of China (30270919, 30330420 and 30471193) and the State Key Basic Research and Development Plan of China (2003CB114302). * Author for correspondence.

Qiu-Hong PAN et al.: Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

nitrogen before stored at –80 °C. 1.2 Chemicals Diethylaminoethyl (DEAE)-Sepharose Fast Flow (FF), carboxymethyl (CM)-sepharose CL-6B, concanavalin A (ConA)-sepharose 4B, sephacryl s-300HR and cyanogen bromide (CNBr)-activated sepharose 4B columns were purchased from Amersham-Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). All other chemicals were purchased from Sigma (St Louis, MO, USA) unless noted otherwise. Rabbit IgG antibody of the anti-carrot cell wall acid invertase was a generous gift from Dr Arnd Sturm (Lurière et al. 1988). 1.3 Enzyme extraction Invertase was extracted essentially according to the method described by Zhang et al. (2001) with modifications, in that the ratio of cold buffer A to apple tissue was 2:1 (v/w). Buffer A medium was composed of 150 mmol/L Tris-HCl (pH 8.0), 2 mmol/L EDTA, 10 mmol/L MgCl 2, 0.2% (v/v) 2-mercaptoethanol, 0.1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 1 mmol/L benzamidine, 10 mmol/L ascorbic acid, and 3% (w/v) polyvinylpolypyrrolidone (PVPP). The slurry was passed through four layers of cheesecloth. The filtrate was centrifuged at 16 000g for 20 min and the supernatant was used for the preparation of the SAI. The residue, which was used for the preparation of CWI, was rinsed with the same buffer without PVPP until the effluent was free of proteins. From this material, CWI was extracted in buffer A containing 0.5 mol/L NaCl at 4 °C with gentle shaking for 24 h. After centrifugation, the supernatant was used for purification of CWI. 1.4 Purification procedures All steps were performed at 4 °C. 1.4.1 Ammonium sulfate precipitation Solid ammonium sulfate was slowly added to the extract up to 90% saturation with gentle stirring and then left overnight. The precipitate formed was collected by centrifugation and dissolved in a minimal volume of buffer B, which was composed of 10 mmol/L Tris-phosphate (pH 6.7), 2 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), 1 mmol/L benzamidine, and 0.1 mmol/L

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phenylmethylsulphonyl fluoride (PMSF), which gave the SAI sample. The suspension was dialysed exhaustively against buffer B and subsequently clarified by centrifugation. For the CWI sample, the precipitate was dissolved in buffer C, containing 20 mmol/L sodium phosphate buffer (pH 7.5), 5 mmol/L EDTA, 1 mmol/L DTT, 0.1 mmol/L PMSF, and 1 mmol/L benzamidine, and dialysed three times against the same buffer. 1.4.2 Ion exchange chromatography The clarified and dialysed SAI preparation obtained from the first step was applied to a DEAE-sepharose FF column (1.6 cm×17.0 cm) that had been equilibrated thoroughly with buffer B. The column was then washed with 10 bed volumes of that buffer and eluted with a linear gradient of 0–500 mmol/L NaCl (pH 6.7) in a total volume of 300 mL at a flow rate of 0.8 mL/min. For CWI, the clarified preparation from the first step was applied to a CM-Sepharose CL-6B column equilibrated and washed with buffer C and eluted with buffer C containing 0–500 mmol/L NaCl. 1.4.3 Gel filtration The invertase-active fractions obtained from ion-exchange chromatography were pooled and concentrated by ultrafiltration (cut-off 8– 10 kDa) to a volume of 3.0 mL. This protein sample was then loaded onto a Sephacryl s-300 column (1.6 cm×75.0 cm) previously equilibrated with buffer D, containing 50 mmol/L sodium acetate buffer (pH 6.0), 100 mmol/L NaCl, and 0.1 mmol/L DTT. The column was eluted with the same buffer at a flow rate of 0.25 mL/min. 1.4.4 Affinity chromatography The fractions of peak activity from the Sephacryl s-300 column were combined, concentrated by ultrafiltration (cut-off 8– 10 kDa) three times against buffer E, which was composed of 100 mmol/L sodium acetate buffer (pH 6.0), 1 mol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L MnCl2, and 1 mmol/L MgCl2, and then applied to a ConA-sepharose 4B column (1.0×17 cm) that had been equilibrated with buffer E. The column was washed extensively with the equilibrating buffer. The protein on the column was subsequently eluted with 50 mL of 100 mmol/L methyl-α-D-mannopyranoside in buffer E. The eluate with

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Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica) Vol. 47 No. 1 2005

invertase activity was concentrated by ultrafiltration (cut-off 8–10 kDa) against 10 mmol/L sodium acetate buffer (pH 6.0) and stored in 0.5 mL aliquots at –80 °C until further use. 1.5 Enzyme assay Acid invertase activities were assayed in the soluble and insoluble fractions in 0.3 mL of 100 mmol/L sodium acetate buffer (pH 4.8), 0.1 mL of 100 mmol/L sucrose, and 0.1 mL enzyme sample, as described by Schaffer et al. (1987). The reducing sugars produced were determined with 3,5-dinitrosalicylic acid according to Miller (1959). One unit of acid invertase is defined as the amount of the enzyme producing 1 µmol glucose per minute under the assay conditions. 1.6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the methods of Laemmli (1970) to determine subunit molecular mass and purity of the protein. The gel was 0.75 mm thick and contained 12% polyacrylamide. Proteins were visualized by silver staining. 1.7 Molecular mass measurement The molecular mass of native acid invertase was measured using a sephacryl s-300 column (1.6 cm×75.0 cm) equilibrated with buffer D. 1.8 Sequencing The sequencing process was performed essentially according to the method described by van den Ende et al. (2001). Briefly, the SDS-PAGE protein bands exhibiting acid invertase activities were subjected to assay on quadrupole-time of flight (Q-TOF2) mass spectrometry/mass spectroscopy (Micromass, Wythenshawe, UK) after digestion by trypsin. 1.9 Effect of fructose The reaction mixtures contained 50 µL enzyme, 50 µL different concentrations of fructose (0, 0.5, 1.0, 1.5, or 2.0 mol/L), 50 µL different concentrations of sucrose (0, 25.00, 31.25, 62.50, 125.00, or 250.00 mmol/L), and 100 µL sodium acetate buffer (pH 4.8). Incubations were performed at 37 °C for 30 min. The fructose produced was determined according to the method of

Jörgensen and Andersen (1973) via glucose oxidase. 1.10 Antibody production Rabbits were injected with the purified SAI (400 µg protein) or CWI (250 µg protein) emulsified in complete Freund’s adjuvant. Four booster injections were given with purified proteins emulsified in incomplete adjuvant at 10 d intervals (150 µg each protein per injection). The antiserum was collected 10 d after the last injection. The IgG fraction was purified by precipitation with 50% saturation (NH4)2SO4 solution and DEAE-Sepharose FF column chromatography. The IgG fraction was then passed over a horseradish peroxidase (POD)-sepharose column to remove antibodies that bound to glycan groups. The POD was bound to CNBr-activated sepharose 4B according to the method of Kenney and Fowell (1992). The POD-sepharose column was equilibrated with 10.0 mmol/L phosphatebuffered saline (PBS; pH 7.5). Purified IgG was slowly passed through this column. The IgG raised against polypeptides of invertase was eluted with the same buffer and dialysed three times against double-distilled (dd) H2O. The dialysed sample then was freeze-dried. The IgG that bound to the glycan group resided on the column and was eluted with 0.1 mol/L glycine-HCl (pH 2.6) and neutralized immediately with a buffer of 1 mol/ L Tris-HCl (pH 8.0). 1.11 Immunoblotting assay Immunoblotting was performed essentially according to the method described by Zhang et al. (2001). Antibody specificity was tested by immunoblotting. 1.12 Immunoinhibition Acid invertase containing 0.02 EU was treated with 0–60 µg anti-SAI antibody or anti-CWI antibody expressed as protein content. Reaction mixtures consisted of 75 µL enzyme and 0–60 µL antibody solution, 50 mmol/L Tris-HCl (pH 7.5), and 150 mmol/L NaCl to a total volume of 150 µL. After incubation at 4 °C for 3 or 24 h, the mixtures were centrifuged at 16 000g for 10 min. The supernatant (100 µL) was used to measure residual acid invertase activity. A reaction mixture containing 0 µL antibody was taken as a control, to which rabbit pre-immune serum (60 µg) or bovine


serum albumin (BSA; 60 µg) was added.

2

Results and Discussion

2.1 Purification, molecular mass, and characteristic sequences Purification procedures of SAI and CWI are summarized in Tables 1 and 2. A comparison of the specific activity of the purified acid invertase with that of crude protein indicated an enrichment of 280-fold for SAI and 35-fold for CWI after the successive purification procedures (Tables 1, 2). The purification procedures were similar for the two classes of acid invertase, Table 1

except for the ion-exchange chromatography step, where anion-exchange chromatography was used for SAI and cation-exchange chromatography was used for CWI. In fact, SAI, having a pI of 4.5–5.0, is negatively charged at pH 6.7, which was the pH of the buffer used for binding to the anion-exchange chromatography medium, whereas CWI, having a pI of approximately 9.0, is positively charged at pH 7.5, the pH of the buffer used for binding to the cation-exchange chromatography medium. The elution profile of acid invertases from ion-exchange chromatography column is shown in Fig. 1.

Purification protocol of soluble acid invertase from apple fruit

Total protein (mg) Crude extract 800.00 650.00 90% (NH4) 2SO4 precipitation DEAE-sepharose FF 17.25 Sephacryl s-300 HR 6.00 ConA-sepharose CL-4B 0.23 Step

Table 2

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Total activity (units) 405.00 347.49 293.01 115.92 32.26

Specific activity Ratio of recoRatio of recovered Purification (units/mg protein) vered protein (%) activity (%) (-fold) 0.500 100.00 100.00 1.00 0.534 81.25 86.87 1.07 16.99 2.16 73.25 34.91 18.32 0.75 28.98 36.64 140.24 0.03 8.07 280.40

Purification protocol of cell wall-bound acid invertase from apple fruit

Total protein (mg) Crude extract 240.00 198.03 90% (NH4) 2SO4 precipitation CM-sepharose 6B 15.91 Sephacryl s-300 HR 4.33 ConA-sepharose CL-4B 0.31 Step

Total activity (units) 187.08 151.30 35.09 31.56 8.64

Specific activity Ratio of recoRatio of recovered Purification (units/mg protein) vered protein (%) activity (%) (-fold) 0.783 100.00 100.00 1.000 0.763 82.63 80.60 0.974 2.206 6.63 72.25 2.820 7.289 1.80 18.68 9.310 27.870 0.13 4.60 35.590

Fig. 1. (a) DEAE-sepharose FF anion-exchange chromatography of soluble acid invertase (SAI; 7.0 mL/fraction) and (b) CM-sepharose CL-4B cation-exchange chromatography of cell wall-bound acid invertase (CWI; 4.0 mL/fraction).

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Upon elution with a total volume of 300 mL of a linear NaCl gradient from 0 to 500 mmol/L, a single peak of SAI activity appeared at approximately 200 mmol/L NaCl (Fig. 1a), whereas two peaks of CWI were observed at approximately 280 and 360 mmol/L (Fig. 1b). However, when the pooled fractions of the two peaks of CWI from the CM-sepharose column were subjected to gel filtration on sephacryl s-300 and then to ConAsepharose, invertase activity was eluted as a single peak in both cases (data not shown). The results are similar to those of the purification of acid invertase from the barley elongating stem tissue (Karuppiah et al. 1989). The two proteins, SAI and CWI, obtained after the Con A-sepharose step both gave a main band with an estimated molecular mass of 50 kDa on denaturing SDS-polyacrylamide gel by silver staining (Fig. 2a). The two proteins could also strongly cross-react with antibody directed against the carrot cell wall invertase (Fig. 2b) and with that against baker’s yeast cell wall invertase (data not shown). The purified SAI and CWI showed a similar apparent molecular mass of 220 kDa,

Fig. 2. Silver-stained sodium dodecyl sulfate-polyacrylamide gel (a) and immunoblotting assay (b) of the purified cell wall-bound acid invertase (CWI) and soluble acid invertase (SAI) from apple fruit. Lane 1, purified CWI (2 µg); lane 2, purified SAI (0.5 µg); lanes 3, 4, immunoblotting of the purified CWI and SAI, respectively, with antibodies directed against carrot cell wall invertase. Mr, molecular mass standards. The arrow indicates the position of apple fruit acid invertases that migrated with an apparent molecular mass of approximately 50 kDa.

determined by gel filtration. The molecular massed of 50 kDa as a subunit and 220 kDa as the holoenzyme are approximately situated in the range of the molecular mass of most acid invertases in plants (Quick and Schaffer 1996). According to the available genomic data, the N-terminal sequences of acid invertases vary with plant species. So, we sequenced the tryptic fragments of the purified proteins by Q-TOF analysis. Among numbers of the sequenced fragments, a characteristic sequence of SAI, GMWECV, was found in the purified SAI and two characteristic sequences of acid invertases, NDPNG and WECP, were found in the purified CWI. This indicates that the purified proteins were two isoforms of acid invertases. 2.2 Enzyme properties The two purified acid invertases showed similar enzymologic properties. The purified SAI and CWI could cleave sucrose and raffinose, but they had much higher affinity for sucrose (Km=2.9 mmol/L for SAI and 7.1 mmol/L for CWI) than for raffinose (Km=562 mmol/L for SAI and 368 mmol/L for CWI). No hydrolytic activity was detected when using stachyose, melezitose, turanose, or trehalose as a substrate. Analysis of the metabolic products by HPLC when using sucrose as a substrate detected only fructose, glucose, and a trace of residual sucrose, but did not detect kestose or neokestose, indicating that there were no sucrosesucrose 1-fructosyltransferase or sucrose-fructan 6fructosyltransferase activities in the purified acid invertases. This also indicates non-contamination by fructan exohydrolase in the purified enzymes. Analysis of the metabolic products by HPLC when using the above-mentioned potential substrates other than sucrose and raffinose detected only non-metabolized substrates in the reaction medium. These data indicate that the preferred substrate of both enzymes is sucrose. This substrate specificity, together with the above-mentioned characteristic sequences and immunoreaction of the purified fractions with antibody against carrot cell wall invertase, demonstrates that the purified enzymes are, indeed, apple fruit acid invertases. The Km values of


the SAI and CWI for raffinose are extremely high at pH 4.8, more than 50-fold higher for CWI and 180fold for SAI than the Km for sucrose, which is distinct from those in other plants, such as melon fruit (Iwatsubo et al. 1992), tomato fruit (Konno et al. 1993), Oryza sativa (Isla et al. 1995), and Solanum tuberosum tubers (Isla et al. 1999), in which the Km value of acid invertase for raffinose was shown to be one- to 20-fold higher than that for sucrose. Conversely, most of the acid invertases from many other plants or tissues can cleave stachyose possessing α-D-fructofuranoside at a low rate (Sampietro et al. 1980; Miller and Ranwala 1994; Isla et al. 1995, 1999), so the fact there was no detectable stachyose cleavage activity of the purified SAI and CWI from apple fruit indicates that apple fruit acid invertases are more specific for sucrose. The effect of fructose on the purified SAI activity was also assayed. Figure 3 shows the characteristics of the fructose inhibition. The intersection of double reciprocal plots of initial velocity against sucrose concentration in the presence of various concentrations of fructose did not occur on either the of 1/Vi axis or the 1/[S] axis and the replot of slopes versus fructose concentration gave a sigmoidal curve, which indicates that the fructose inhibition has neither a simple competitive nor simply non-competitive pattern (Fig. 3). The inhibition of the purified CWI by fructose showed essentially the same pattern (data not shown) as that for the SAI. Therefore, the fructose inhibition should be a mixed form in this case. This result is markedly different from the situation in other plants, in which the intersection of double reciprocal plots has been shown to occur on the 1/Vi axis with the replot of slopes versus fructose concentration being a sigmoidal curve. These characteristics of the inhibition by fructose were believed to fit in with a complex competitive type through two interacting sites on the invertase (Sampietro et al. 1980; Lopez et al. 1988; Isla et al. 1991). There is also a classical competitive inhibition of invertase by fructose, which was observed in Ricimus communis, where the replot of slopes versus fructose concentration showed

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Fig. 3. Double-reciprocal plot of initial velocity against sucrose concentration. Velocities were determined at fructose concentrations of 0, 100 200, 300, and 400 mmol/L (lanes labeled, respectively, S1, S2, S3, S4, and S5). The inset represents replot of slope against fructose concentrations. Values of slope were those calculated from the double-reciprocal plot of initial velocity against sucrose concentration.

a straight line (Prado et al. 1985). Hence, the inhibition of acid invertase observed in the present study reveals a novel regulatory pattern of the enzyme by fructose. It is of note that we tried to isolated the proteinous inhibitor of acid invertase from the fruit cell walls according to the methods described by Bracho and Whitaker (1990) and Pressey (1994), but we have not obtained any fraction-inhibiting acid invertase activity. So, this proteinous inhibitor may not exist in apple fruit. This result confirms the suggestion made in our previous report (Zhang and Wang 2002). 2.3 Antibody specificity The IgG fractions against the purified SAI and CWI from immunized rabbits were purified from raw antisera by (NH4)2SO4 precipitation and DEAE-sepharose chromatography. These IgG fractions showed two bands, a heavy chain (50 kDa) and a light chain (25 kDa), on SDS-PAGE gel after passing through the DEAE-sepharose column (Fig. 4a, b, lane 3). Because the acid invertases, like those found in other plants (Konno et al. 1993; Ruffner et al. 1995), are glycosylated, as shown above by binding to ConA-

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Fig. 4. Coomassie Brilliant Blue R-250-stained sodium dodecyl sulfate-polyacrylamide gels of IgG fractions obtained at each stage of purification of anti-soluble acid invertase antiserum (a) and anti-cell wall acid invertase antiserum (b). Lane 1, fractions bound to horseradish peroxidase (POD)-sepharose and obtained by elution with 0.1 mol/L glycine-HCl (pH 2.6); lane 2, fractions that could not be bound to POD-Sepharose; lane 3, fractions obtained after DEAE-sepharose chromatography; lane 4, (NH4)2SO4 precipitation resuspentionate. M, standard protein molecular mass markers.

sepharose and by a blotting assay with periodic acidSchiff giving a positive result (data not shown), the antibodies raised against the glycan groups of the enzymes may cross-react with other glycoproteins rather than acid invertases, thus resulting in non-specific immunoreactivity. To remove antibodies cross-reacting with glycans, the IgG fractions of these antisera were passed through a POD-linked sepharose column to which the antibodies specific to the glycan groups of the enzymes were bound by affinity through POD, a glycoprotein, whereas the antibodies reactive with the acid inveratse polypeptides could not be retained. Figure 4a,b (lanes 1, 2) shows the migration of the IgG fractions on an SDS-PAGE gel, which indicates practically the same migrating rate of the fraction bound to the affinity column (Fig. 4a,b, lane 1) as that of the fraction that was not bound to the column (Fig. 4a, b, lane 2). The antibodies against both SAI and CWI obtained after the purification on a DEAE-sepharose column were shown by immunoblotting to be able to react with

both of the fruit acid invertases (Fig. 5a, c, lanes 1–4), but they could also cross-react with the heavily glycosylated plant protein horseradish peroxidase (Fig. 5a, c, lane 5). The anti-SAI and anti-CWI antibodies purified on the POD-sepharose column were shown to immunoreact with both fruit acid invertases (Fig. 4b, d, lanes 1–4), but they did not cross-react with the horseradish peroxidase (Fig. 5b, d, lane 5). These results provide further proof of the glycoprotein nature of the two apple fruit acid invertases, of which the glugans can be an effective antigen to affect the immunospecificity of the raised antibodies and, conversely, to reveal the specificity of the antibodies purified against the protein part of the acid invertases. It is of note that each major band with an apparent molecular mass of 50 kDa obtained at the various stages of the purification was clearly detected by immunoblotting with the purified antibodies (Fig. 5a– d, lanes 1–4), which is coincident with the subunit molecular mass of the purified SAI and CWI (Fig. 2a, b). However, three bands, at 68, 50, and 30 kDa, were


Fig. 5. Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels for comparing the specificity of antibodies raised against the acid invertases after purification on DEAE-sepharose with that against the acid invertases obtained after removal of antibodies reactive with glycan groups. Proteins in (a, b) were obtained at each stage of the purification of soluble acid invertase (SAI) and those in (c, d) were obtained at each stage of the purification of cell wall acid invertase (CWI). The proteins were immunorecognized with the anti-SAI IgG obtained after purification on DEAE-sepharose (a), or with anti-SAI IgG that was not bound to the horseradish peroxidase (POD)sepharose (b) or with the anti-CWI IgG obtained after purification on DEAE-sepharose or with anti-CWI IgG that was not bound to the POD-Sepharose. Lanes show immunoblots of the fractions after: (1) ammonium sulphate precipitation; (2) ion-exchange chromatography on DEAEsepharose (for SAI) or CM-sepharose (for CWI); (3) gel filtration on sephycryl s-300; (4) affinity chromatography on ConA-sepharose; and (5) immunoblots of the horseradish peroxidase. The molecular masses of immunoreactive proteins are indicated on the left in kDa.

immunorecognized from the crude proteins obtained after (NH4)2SO4 precipitation of the soluble and cell wall crude homogenate. The same three bands were also immunodetected by the POD-sepharose-purified antibodies in the soluble crude homogenate of leaves and flowers from M. domestica (Fig. 6). Although several types of protease inhibitor, including PMSF, benzamidine, and EDTA, were added into the extracting medium to prevent the enzymes from being degraded, we cannot exclude the possibility that the 68 kDa protein may be an intact glycosylated soluble

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Fig. 6. Immunodetection of acid invertase proteins in the soluble crude extracts from the different organs of apple tree (1, leaves; 2, flower; 3, fruit harvested at 7th day after full bloom) with the anti-apple fruit soluble acid invertase antibody purified by horseradish peroxidase (POD)sepharose column chromatography. Molecular masses of immunoreactive bands (in kDa) are indicated on left of the blot.

or cell wall invertase and the 30 kDa peptide may be a degraded fragment. The 68 and 30 kDa polypeptides were lost during the present purification. However, previous studies in orange fruit (Konno et al. 1993) and tomato fruit (Takehana and Nakagawa 1970) reported similar results, where the authors believed that they had identified several isoforms of acid invertase. In our previous report, the antibody raised against carrot cell wall invertase immunorecognized a polypeptide of 30 kDa from apple fruit (Malus domestica Brohk cv. Golden Delicious; Zhang et al. 2001). The antibody specificity was further assayed by immunoinhibition. When the given buffer system containing 60 µg antibody (expressed as protein content) and 0.02 EU SAI was incubated for 3 or 24 h at 4 °C, the enzyme activities were inhibited by 59.0% and 87.9%, respectively, by the anti-SAI antibody (Fig. 7a) and by 61.9% and 94.1%, respectively, by the antiCWI antibody (Fig. 7b). The antibodies raised against SAI and CWI produced a similar in vitro inhibitory

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Schaffer 1996; Isla et al. 1999; Zhang et al. 2001). This assessment was supported in the present study. The anti-SAI antibody could recognize CWI (data not shown) and anti-CWI antibody could also cross-react with the SAI of apple fruit (Fig. 7). This mutual crossreaction of the antibodies with the corresponding antigens and their similarity in terms of the in vitro inhibitory pattern on SAI (Fig. 7) suggest that the peptidic moiety is identical. References Beruter J (1985). Sugar accumulation and changes in the activities of related enzymes during development of apple fruit. J Plant Physiol 121, 331–334. Beruter J, Studer FME, Ruedi P (1997). Sorbitol and sucrose partitioning in the growing apple fruit. J Plant Physiol 151, 269–276. Bracho GE, Whitaker JR (1990). Purification and partial characterization of potato invertase and its endogenous proteinaceous inhibitor. Plant Physiol 92, 386–394. Fahrendorf T, Beck E (1990). Cytosolic and cell wall acid inver-

Fig. 7. Immunoinhibition of the soluble acid invertase of apple fruit by the anti-soluble acid invertase antibody (a) and anti-cell wall acid invertase antibody (b). The relative activity of acid invertase was calculated in relation to the acid invertase activity measured in the medium containing no antibody (0 µg antibody but 60 µg pre-immune serum or 60 µg bovine serum albumin). The acid invertase activity assayed in the antibody free medium was taken as 100%.

tases from leaves of Urtica dioica L: A comparison. Planta 180, 237–244. Isla MI, Vattuone MA, Sampietro AR (1991). Modulation of potato invertase activity by fructose. Phytochemistry 30, 425–426. Isla MI, Salerno G, Pontis H, Vattuone MA, Sampietro AR (1995). Purification and properties of the soluble acid invertase from Oryza sativa. Phytochemistry 38, 321–325.

pattern on soluble acid invertase (Fig. 7a, b). The immunoinhibition assay of the CWI activities by the two antibodies gave substantially the same results (data not shown). These results confirm those of the immunoblotting assay mentioned above, further demonstrating the specificity of the antibodies produced in the present study. As reported previously in other plants, SAI and CWI may be the products of the same gene family and, so, exhibit a high degree of antigenic homology, allowing for the interchange of antibodies from one plant species to the other, and the antisera against the two invertases can recognize the antigens mutually (Lauriere et al. 1988; Sturm and Chrispeels 1990; Quick and

Isla MI, Vattuone MA, Ordonez RM, Sampietro AR (1999). Invertase activity associated with the walls of Solanum tuberosum tubers. Phytochemistry 50, 525–534. Iwatsubo T, Nakagawa H, Ogura H, Hirabayashi T, Sato T (1992). Acid invertase of melon fruits: Immunochemical detection of acid invertases. Plant Cell Physiol 33, 1127–1133. Jörgensen OT, Andersen B (1973). An improved glucoseoxidaseperoxidase-coupled assay for β-fructofuranosidase activity. Anal Biochem 53, 141–145. Karuppiah N, Vadlamudi B, Kaufman PB (1989). Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley elongating stem tissue. Plant Physiol 19, 993–998. Kenney A, Fowell S (1992). Some alternative coupling

Qiu-Hong PAN et al.: Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit chemistries for affinity chromatography: Practical protein chromatography. Methods Mol Biol 11, 173–196. Klann EM, Hall B, Bennett AB (1996). Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiol 112, 1321–1330. Konno Y, Vedvick T, Fitzmaiurice L, Mirkor JE (1993).

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