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Localization of the Fe(III)-Zn(II) Purple Acid Phosphatase in Dry Kidney Beans Using Immunofluorescence and Immunogold Electron Microscopy
Annals of Botany 82 : 235–241, 1998
Localization of the Fe(III)-Zn(II) Purple Acid Phosphatase in Dry Kidney Beans Using Immunofluorescence and Immunogold Electron Microscopy M. G R O TE*, C. S C H W E R D T F E G ER†, R. W I E R M A NN†‡, R. R E I C H E L T* and H. W I T Z EL§ * Institut fuX r Medizinische Physik und Biophysik, † Institut fuX r Botanik and § Institut fuX r Biochemie, WestfaX lische Wilhelms-UniersitaX t, MuX nster, Germany Received : 10 March 1998
Returned for revision : 26 March 1998
Accepted : 30 April 1998
Localization of purple acid phosphatase (PAP) from the seeds of kidney beans, Phaseolus ulgaris (L.), was performed using light and transmission electron microscopy. After rehydration and aqueous fixation, cryo-sections of bean cotyledon tissue showed a bright immunofluorescent signal in the cytoplasm of cells whereas cell walls and reserve materials (starch, protein bodies) remained unstained. In ultrathin sections of dry cotyledon tissue anhydrously fixed in acrolein vapour and embedded in Lowicryl resin, PAP mapped exclusively to ribosome-rich areas of the cytoplasm. In view of these results, we propose that kidney bean PAP might possibly be engaged in mechanisms involved in the triggering of seed dormancy. # 1998 Annals of Botany Company Key words : Phaseolus ulgaris (L.), kidney bean, purple acid phosphatase, immunofluorescence microscopy, immunogold electron microscopy, anhydrous vapour fixation, acrolein.
INTRODUCTION Acid phosphatases are ubiquitously distributed in plants, and occur in numerous tissues. Several classes of acid phosphatases from plant sources have been reported (Fujumoto, Nakagawa and Ohara, 1977 ; Beck et al., 1986 ; Ullah and Gibson, 1988 ; LeBansky, McKnight and Griffing, 1992 ; Ferte et al., 1993 ; Cashikar, Kumaresan and Rao, 1997 ; for review see Vincent, Crowder and Averill, 1992 ; Duff, Sarath and Plaxton, 1994). An intensely studied plant enzyme is purple acid phosphatase (PAP) from the dry seeds of kidney beans (Phaseolus ulgaris L.). Kidney bean (kb)PAP was purified to homogeneity and biochemically and biophysically characterized in detail (Beck et al., 1986 ; Ullah and Gibson, 1988 ; Suerbaum et al., 1993 ; Klabunde et al., 1994, 1995 ; Stahl et al., 1994 ; Stra$ ter et al., 1995). The enzyme was also sequenced (Klabunde et al., 1994) and crystallized (Stra$ ter et al., 1992). PAPs contain a dimetal centre in the active site (Doi, Antanaitis and Aisen, 1988 ; Vincent, Olivier-Lilley and Averill, 1990). PAP from kidney beans is a homodimeric Fe(III)-Zn(II) metalloenzyme with a molecular mass of 111 kD (Beck et al., 1986, 1988 a, b). By X-ray structure analysis the ligands for the two metal ions could be elucidated (Stra$ ter et al., 1995 ; Klabunde et al., 1996). Kidney bean PAP catalyses the hydrolysis of activated phosphoric acid esters and anhydrides like ATP at a pH range from 4 to 7. Disregarding the first 122 amino acids of kidney bean PAP N-terminus, its sequence shows less than 20 % similarity to uteroferrin, a purple acid phosphatase of mammalian origin (porcine uterus). In this connection it is ‡ For correspondence, E-mail Wierman!uni-muenster.de
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surprising that the ligands of the two metal ions are conserved so that the Zn(II) could be exchanged against Fe(II) without any loss of catalytic activity. Electron paramagnetic resonance (EPR) (Dietrich et al., 1991) and Mo$ ssbauer data (Suerbaum et al., 1993) of the Zn(II) exchanged Fe(III)-Fe(II) enzyme from kidney beans are nearly identical to those of the mammalian enzyme. The plant and the mammalian enzymes are possibly derived from a common ancestral enzyme. During evolution, PAP might have taken over different functions in plant and mammalian lines. Despite this wealth of structural information on kidney bean PAP, there are few data concerning the precise location and the physiological role of this enzyme. Klabunde et al. (1995) suggested an anti-oxidant function for the enzyme from kidney beans. Using enzyme cytochemistry by phosphatase activity staining, Cashikar et al. (1997) localized kbPAP in the cell walls of the peripheral cotyledon cells and argued that the enzyme might play a role in the nutrition of the developing embryo by mobilizing organic phosphate in the soil. In order to obtain more insight into the biological function of the enzyme, its precise cellular and subcellular location have to be established. Since most structural studies on kbPAP were performed on the enzyme extracted from the dry seed we chose to start here with our localization study. We used highly specific antibodies against kbPAP in combination with two independent immunomicroscopic techniques : immunofluorescence light microscopy for a histological survey, and immunogold electron microscopy for ultrastructural analysis. For the immunofluorescent approach, specimens were treated in the conventional way using aqueous fixation of rehydrated beans. However, for electron microscopy, dry plant tissue can be prepared in an anhydrous way, by, e.g. osmium vapour fixation (Hallam,
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
1976 ; Opik, 1980, 1985 ; Smith, 1991) thus preserving the dehydrated morphology of the cells. Since osmium fixation severely impairs antigenicity of most antigens (Griffiths, 1993), we modified this technique using acrolein vapour instead of osmium vapour as the fixative and combined this kind of anhydrous fixation with immunogold microscopy localizing water-soluble allergens in dry grass pollen (Grote et al., 1994).
Seeds of Phaseolus ulgaris (L.), the red kidney bean, were obtained from Mu$ ller’s Mu$ hle (Gelsenkirchen, Germany).
six washes (each lasting 5 min) with TBS containing 0±05 % (v}v) Tween 20, the strips were incubated with an antibodyalkaline phosphatase conjugate (goat anti-mouse IgG ; Atlanta, Heidelberg, Germany, No. S3721), diluted by the factor of 10% with blocking solution for 1 h. After six further washes [five with TBS containing 0±05 % (v}v) Tween 20 and one with TBS without Tween 20] the strips were incubated without agitation in substrate [66 ml 5 % (w}v) nitro-blue-tetrazolium-chloride, dissolved in 70 % (v}v) dimethylformamide, 33 ml 5 % (w}v) 5-bromo-4-chloro-3indolylphosphate dissolved in dimethylformamide and 9±9 ml 100 m Tris}HCl (pH 9±5) plus 100 m NaCl and 5 m MgCl ]. The reaction was terminated after 5–30 min # and strips were washed three times with water.
Enzyme preparation
Epifluorescence microscopy
Isolation and purification of the enzyme from dry seeds followed the procedure described by Klabunde et al. (1994) for determination of the amino acid sequence. 30 mg of pure enzyme can be obtained from 300 g bean meal after an extraction step, ethanol precipitation, ethanol fractionation, ammonium sulphate precipitation and finally an affinity chromatography step on Blue sepharose CL-4B.
Fixation and preparation of the freeze-sections. Dry seeds of Phaseolus ulgaris (L.) were soaked in water for 20 h before the red hull was removed and the cotyledons were cut into cubes of approx. 3–4 mm square. These tissue blocks were fixed in 4 % para-formaldehyde (w}v) in 50 m phosphate buffer, pH 7±2 for 4 h at room temperature. After fixation, the specimens were washed four times (each time for 10 min) in phosphate-buffered saline (PBS) buffer (10 m sodium phosphate buffer, 150 m NaCl), pH 7±4. Then they were embedded in freezing medium (Reichert and Jung, Heidelberg, Germany) and cut into 20 µm thick sections using a cryo-microtome (Frigocut 2700, Reichert and Jung, Heidelberg, Germany). Immunolabelling. In situ localization of purple acid phosphatase was performed following the method of Scha$ chtele and Steup (1986). Frozen-sections of bean cotyledon tissue were placed on slides. The thawed sections were washed three times (10 min each) with PBS buffer containing 0±5 % BSA, pH 7±4 at ambient temperature then incubated with the following solutions : (1) anti-purple phosphatase antibody, diluted 1 : 1600 in PBS, 1 h ; (2) washing in PBS, three¬10 min each ; (3) goat anti-mouse IgG-FITC (fluorescein isothiocyanate) conjugate diluted 1 : 125 in PBS, 1 h ; (4) washing in PBS, three¬10 min. Control sections were : (1) incubated with mouse preimmune antibodies instead of the specific antibodies ; and (2) the primary antibody was omitted and the sections incubated with the IgG-FITC conjugate alone. In situ localization by epifluorescence microscopy. After immunolabelling, sections were analysed by epifluorescence microscopy using a Leitz Diaplan fluorescence microscope (Leitz, Wetzlar, Germany) with filter blocks L3 and GR. Epifluorescence micrographs were recorded on Fujichrome 400 film.
MATERIALS AND METHODS Plant material
Preparation of crude extracts for antibody testing All steps of extraction were performed between 0–4 °C. 3 g of cotyledon tissue were homogenized in 5 ml TBS (50 m Tris}HCl, 150 m NaCl, 1 m MgCl , pH 7±8). The # extract was filtered through four layers of nylon gauze and subsequently centrifuged in a Sorvall SS-34 rotor at 10 000 rpm for 10 min. The supernatant was mixed with sample buffer and used for electrophoresis. Antibody preparation Four-week-old female Balb c mice were immunized according to Weiler (1986). Each injection contained 20 mg of purified kidney bean purple acid phosphatase in incomplete Freund’s adjuvant. Primary injections were given intraperitoneally at day 1 and day 7. Further booster injections were given every 4 weeks. Preimmune serum was collected before first priming. 100–200 ml serum samples were collected 1 and 2 weeks after each booster injection by tail vein bleeding and pooled for experimental use. SDS-PAGE and immunoblotting SDS-PAGE was performed according to Laemmli (1970). The sample was incubated at 95 °C for 5 min and 50 mg protein was applied per cm gel. It was run at 15 mA in stacking gel and 20 mA in separation gel at room temperature. After transfer of the separated proteins onto nitrocellulose (Towbin, Staehlin and Gordon, 1979), the blot was cut into strips for probing with antibodies. First, the strips were incubated for 1 h with TBS containing 0±05 % (v}v) Tween 20 and 3 % (w}v) powdered milk (blocking solution) under constant agitation. The solution was then substituted by serum diluted by the factor of 5¬10$ with blocking solution and incubated for 1 h. After
Electron microscopy Anhydrous fixation. To gain information from the original dehydrated state of the specimen, fixation and preparation techniques for electron microscopy were kept strictly anhydrous. Since preliminary experiments showed that anhydrous fixation in paraformaldehyde vapour (a mild form of fixation preserving antigenicty of most antigens ; Griffith, 1993) rendered unsectionable specimens, fixation
Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans was performed in acrolein vapour (Grote et al., 1994). After removing the seed shell, cubes of approx. 1 mm$ were excised with a sharp razor blade from the peripheral part of the cotyledons and placed on cover slips. The cover slips were put into a desiccator together with a Petri dish filled with acrolein liquid (Riedel-de-Haen, Seelze, Germany). The desiccator was tightly sealed and placed into a fume hood at ambient temperature. Samples stayed in acrolein vapour for 13 d since preliminary experiments showed that shorter periods of fixation tended to result in unsectionable specimens. Dehydration and embedding. After vapour fixation, samples were completely dehydrated in dimethoxypropane (DMP) and absolute ethanol for 4 h each and then transferred successively into four ethanol : Lowicryl K4M mixtures (2 : 1, overnight ; 1 : 1, 8 h ; 1 : 2, overnight ; 1 : 5, 8 h). Afterwards, they were placed into pure Lowicryl resin where they remained for 48 h with two changes of the medium at room temperature. Polymerization was carried out at ®35 °C after the samples had been placed in fresh resin and slowly cooled over a period of 4 h to ®35 °C before UV irradiation was started. Sectioning. Ultrathin sections were cut on a Reichert OM U3 ultramicrotome and placed on nickel grids for immunolabelling procedures. Immunolabelling. Ultrathin sections on nickel grids (room temperature, moist chamber, PBS buffer at pH 7±4, TrisBSA buffer at pH 8±2) were put on droplets of the following solutions : (1) 5 % BSA in PBS, 15 min ; (2) mouse antipurple acid phosphatase antibody, diluted 1 : 2000 in PBS, 2 h ; (3) PBS, two¬5 min and Tris-BSA, 5 min ; (4) goat anti-mouse IgG coupled to 10 nm colloidal gold particles (GAM IgG 10) diluted 1 : 10 in Tris-BSA, 1 h ; (5) Tris-BSA, 5 min and distilled water, two¬5 min. Controls. Controls were performed (1) by replacing the primary specific antibody with mouse preimmune antibodies and (2) by omitting the primary specific antibody and incubating the sections with the gold-conjugate alone. Staining. After immunoabelling, sections were stained for 6 min in uranyl acetate and for 10 sec in lead citrate. Electron microscopy and documentation. After immunocytochemistry and staining, sections were analysed in a Hitachi H500 transmission electron microscope operated at 75 kV. Photographs were taken using Agfa-Gevaert 23D56 film. RESULTS Specificity of antibodies When crude extracts of Phaseolus ulgaris (L.) seeds were blotted onto nitrocellulose and tested with the antibodies, a strong specificity to purple acid phosphatase was shown (Fig. 1). For this reason they were used for immunolabelling experiments.
Immunofluorescence localization of PAP Whereas cell walls are devoid of fluorescent staining, a bright fluorescent signal is given by the cytoplasm in all cells
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F. 1. Immunoblot showing the reactivity of crude extract of dry seeds of Phaseolus ulgaris (L.) with mouse antibodies directed against purple acid phosphatase. Lane a, Crude extract stained with ponceau S ; lane b, crude extract stained with preimmune serum ; lane c, crude extract stained with specific immune serum. (The faint staining below the 52 kD PAP main band most probably results from the fact that the enzyme may occur with variable degrees of glycosylation. This would lead to an electrophoretical separation of the differently glycosylated molecules.)
of the cotyledons with the exception of the vascular tissue (Fig. 2 A and B). However, starch grains and numerous small particles of 2±2–2±5 µm diameter (protein bodies) do not show immunofluorescence. After treating the sections with pre-immune serum, as well as with IgG-FITC conjugate alone, a negligible degree of non-specific background fluorescence can be observed (Fig. 2 C–E). Similar results concerning the localization of purple acid phosphatase were obtained by laser scanning microscopy (data not shown).
Immunogold localization of PAP After incubating ultrathin sections of bean cotyledon tissue with anti-PAP antibodies and gold-conjugated secondary antibodies (Fig. 3 A), gold particles bind specifically to the cytoplasm of the cells but not the cell walls. Within the cytoplasm, protein bodies, amyloplasts (not shown in Fig. 3 A) and mitochondria show almost no decoration with the gold label. However, numerous gold particles are found associated with accumulations of ribosomes, which fill the spaces between the various cell organelles and inclusions of reserve materials throughout the cell. Control sections incubated with the preimmune antibodies revealed a low degree of non-specific staining (Fig. 3 B). Sections incubated with the gold-conjugated secondary antibody alone were almost completely free from gold particles (micrograph not shown).
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
A
B
C
D
E
F. 2. Immunofluorescence localization of PAP in rehydrated seeds of Phaseolus ulgaris (L.). Cross-section of a cotyledon incubated with antipurple acid phosphatase antibodies (A, filter block L3 ; B, filter block GR), with preimmune serum (C, filter block L3 ; D, filter block GR) or with IgG-FITC conjugate alone (E, filter block GR). Bars ¯ 10 µm (A, C–E, ¬416 ; b, ¬1090).
DISCUSSION In the present study two independent microscopic techniques were used to localize PAP in kidney beans : immunofluorescence microscopy and immunogold electron microscopy. Immunofluorescence microscopy using cryo-
sections of rehydrated and aqueously fixed kidney beans revealed positive staining throughout the cytoplasmic matrix of the cotyledon cells. Neither cell walls nor reserve materials (starch, protein bodies) yielded specific fluorescence. This comparatively coarse screening was supplemented and specified by transmission electron mi-
Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
239
A
B F. 3. A, Immunoelectron microscopic localization of PAP in dry seeds of Phaseolus ulgaris (L.). Detail from cotyledon cell showing part of cell wall (W) and cytoplasm with mitochondria (M), accumulation of ribosomes (R) and protein bodies (P) after incubation with mouse antipurple acid phosphatase antibodies followed by 10 nm colloidal gold conjugated secondary antibodies. Gold particles predominantly map to ribosome-rich areas in the cytoplasm. Bar ¯ 0±2 µm. B, Immunoelectron microscopic control experiment. Detail of similar cell compartment as in A after incubation with mouse preimmune antibodies followed by 10 nm colloidal gold-conjugated secondary antibodies. Few non-specifically bound gold particles are observed. Bar ¯ 0±2 µm.
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
croscopy. Since conventionally fixed and embedded dry kidney beans—besides rendering almost unsectionable specimens—do not reflect the original dehydrated state of the cells, we used an alternative fixation technique, i.e. vapour fixation. In a modified form, using acrolein vapour instead of osmium vapour, this technique had been applied in our lab for the immunocytochemical detection of highly water-soluble allergens in grass pollen species (Grote et al., 1994). Applying this anhydrous preparation technique for the first time in the localization of kbPAP eliminated any suspicion of a possible rehydration of the cells which might have been accompanied by alterations of cellular morphology including possible dislocation of the enzyme. Thus the truly desiccated state of the cells was not broken. Immunogold electron microscopy of anhydrously prepared kidney beans mapped PAP to ribosome-rich areas within the cytoplasm of the cotyledon cells. Similar to our light microscopic findings, no labelling was found in the cell walls, starch grains or protein bodies. Though gold particles seem to be located freely in the cytosol, we cannot exclude the possibility that they are confined to membrane-bounded cellular compartments or vesicles. Electron microscopy of dry bean cells, as is true of dehydrated tissues in general, presents a special and somewhat puzzling aspect of cellular morphology if compared with the more ‘ normal ’ look of hydrated cells. Thus, the cytoplasm shows many vacuolar spaces and coils of membranes which are not always easy to interpret and assign to conventional cellular compartments and organelles. For the two immunohistochemical approaches used, pretreatment of the plant material was not identical. For light microscopic experiments, beans were rehydrated and aqueously fixed, whereas electron microscopy was performed on dry tissue. Thus, strictly speaking, the results of the two techniques might not be considered comparable. However, both immunofluorescence and immunoelectron microscopy yielded labelling patterns which are fully compatible. Gross dislocations such as migration of PAP from the cytoplasm into the cell walls which would have been visible under the light microscope did not take place during the short initial rehydration period. This conclusion must be drawn from the results of electron microscopy which represent the labelling status of the original dry kidney bean cell. In the plant tissues studied to date (Giordani, 1978 ; Matile, 1978 ; Giordani et al., 1986 ; Ferte et al., 1993 ; Duff et al., 1994) acid phosphatases were generally localized in a number of different cell compartments such as vacuoles, cytosol and cell walls. Extracellular phosphatases appear to be ubiquitous in roots and plant cell cultures (Ferte et al., 1993 ; Duff et al., 1994). In laticifers acid phosphatases were located in the lumen of provacuoles and vacuolar apparatus (Giordani et al., 1986). The authors suggested that these enzymes are involved in the turnover of cytoplasmic components (cf. Matile, 1978). Concerning the localization of kidney bean PAP, a recent report (Cashikar et al., 1997) found the enzyme exclusively in the walls of the peripheral cotyledon cells. These authors applied a histochemical procedure which uses ATP as substrate for PAP : free phosphate released on PAP catalysis was precipitated as
insoluble lead phosphate. This technique, however, does not distinguish between different enzymes ; it would simply indicate any ATP-hydrolysing system. From their cytochemical observations, Cashikar et al. (1997) hypothesized that kidney bean PAP is involved in the mobilization of organic phosphates in the soil for the developing embryo. In view of the present immuno-electron microscopic results which show PAP to be associated with intracellular areas rich in ribosomes their hypothesis does not appear plausible. PAPs from human macrophages seem to be involved in killing microbes and the degradation of bone by production of free radicals. This supports the hypothesis that animal PAPs are involved in the formation of species of active oxygen (Suerbaum et al., 1993 ; Hayman and Cox, 1994 ; Klabunde et al., 1995). PAP from kidney beans, however, does not seem to function in such a manner because its Fe(III)-Zn(II) centre cannot form free oxygen radicals. Moreover, its cytosolic localization is not compatible with such a function. Thus, at present, we still cannot define the exact biological function of PAP. Its localization pattern in ribosome-rich areas of the cytosol suggests that the enzyme might be involved in metabolic}catabolic processes whose nature, however, we do not yet understand. Since morphological and cytochemical investigations of the dry seed required specialized techniques, we felt it worthwhile to present our new information on the localization of PAP in dry kidney beans in a separate paper. However, it will be necessary to extend these studies by following the expression of this enzyme in various stages of seed development and germination. The origin and fate of PAP in kidney beans could be followed more closely thus providing additional clues to its biological function. A C K N O W L E D G E M E N TS The work was supported by the Deutsche Forschungsgemeinschaft (to H. W.). The authors appreciate the skilful technical assistance of Mr U. Malkus and the expert photography of Mrs G. Kiefermann. We are very grateful to Professor M. Steup (Institut fu$ r Biochmie und Molekulare Physiologie, Potsdam), and Dr B. Greve (Institut fu$ r Strahlenbiologie, Mu$ nster) for the generous gift of antisera against purple acid phosphatase from kidney beans and the performance of the immunoblot experiment. The research described in this paper was initiated by Prof. Dr. H. Witzel, who made fundamental contributions to the biochemistry and biophysics of purple acid phosphatases. He died on 1 Sept. 1996. LITERATURE CITED Beck JL, McArthur MJ, de Jersey J, Zerner B. 1988 a. Derivatives of the purple phosphatase from red kidney bean : replacement of zinc with other divalent metal ions. Inorganica et Chimica Acta 153 : 39–44. Beck JL, de Jersey J, Zerner B, Hendrich MP, Debrunner PG. 1988 b. Properties of the Fe(II)-Fe(III) derivative of red kidney bean purple phosphatase. Evidence for a binuclear Zn–Fe center in the native enzyme. Journal of the American Chemical Society 110 : 3317–3318.
Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans Beck JL, McConachie LA, Summors AC, Arnold WN, de Jersey J, Zerner B. 1986. Properties of a purple phosphatase from red kidney bean : a zinc-iron metalloenzyme. Biochimica et Biophysica Acta 869 : 61–68. Cashikar AG, Kumaresan R, Rao NM. 1997. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase. Plant Physiology 114 : 907–915. Dietrich M, Mu$ nstermann D, Suerbaum H, Witzel H. 1991. Purple acid phosphatase from bovine spleen. Interactions at the active site in relation to the reaction mechanism. European Journal of Biochemistry 199 : 105–113. Doi K, Antanaitis BC, Aisen P. 1988. The binuclear iron centers of uteroferrin and the purple acid phosphatases. Structure and Bonding 70 : 3–26. Duff SMG, Sarath G, Plaxton WC. 1994. The role of acid phosphatases in plant phosphorus metabolism. Physiologia Plantarum 90 : 791–800. Ferte N, Moustacas A-M, Nari J, Teissere M, Borel M, Thiebart I. 1993. Characterization and kinetic properties of a soya-bean cell-wall phosphatase. European Journal of Biochemistry 211 : 297–304. Fujumoto S, Nakagawa T, Ohara A. 1977. Isolation of a violet-colored acid phosphatase from soybean. Agricultural and Biological Chemistry 41 : 599–600. Giordani R. 1978. Autophagie cellulaire et diffe! renciation des laticife' res non articule! s chez une Ascle! piade. Biologie Cellulaire 33 : 253–260. Giordani R, Nari J, Noat G, Sauve P. 1986. Purification and molecular properties of an acid phosphatase from Asclepias curassaica latex. Plant Science 43 : 207–212. Griffiths G. 1993. Fine structure immunocytochemistry. Berlin : SpringerVerlag. Grote M, Dolecek C, van Ree R, Valenta R. 1994. Immunogold electron microscopic localization of timothy grass (Phleum pratense) pollen major allergens, Phl pI and Phl pV, after anhydrous fixation in acrolein vapor. Journal of Histochemistry and Cytochemistry 42 : 427–431. Hallam ND. 1976. Anhydrous fixation of dry plant tissue using nonaqueous fixatives. Journal of Microscopy 106 : 337–342. Hayman AR, Cox TM. 1994. Purple acid phosphatase of the human macrophage and osteoclast. Journal of Biological Chemistry 269 : 1294–1300. Klabunde T, Stra$ ter N, Krebs B, Witzel H. 1995. Structural relationship between the mammalian Fe(III)-Fe(II) and the Fe(III)-Zn(II) plant purple acid phosphatases. Federation of European Biochemical Societies Letters 367 : 56–60. Klabunde T, Stra$ ter N, Fro$ hlich R, Witzel H, Krebs B. 1996. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures. Journal of Molecular Biology 259 : 737–748. Klabunde T, Stahl B, Suerbaum H, Hahner S, Karas M, Hillenkamp F, Krebs B, Witzel H. 1994. The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrixassisted laser desorption}ionozation mass spectrometry and
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automated Edman sequencing. European Journal of Biochemistry 226 : 369–375. Laemmli UK. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227 : 680–685. LeBansky BR, McKnight TD, Griffing LR. 1992. Purification and characterization of a secreted purple phosphatase from soybean suspension cultures. Plant Physiology 99 : 391–395. Matile P. 1978. Biochemistry and function of vacuoles. Annual Reiew of Plant Physiology 29 : 193–213. Opik H. 1980. The ultrastructure of coleoptile cells in dry rice (Oryza satia L.) grains after anhydrous fixation with osmium tetroxide vapor. New Phytologist 85 : 521–529. Opik H. 1985. The fine structure of some dry seed tissues observed after completely anhydrous chemical fixation. Annals of Botany 56 : 453–466. Scha$ chtele C, Steup M. 1986. α-1.4 Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.). In situ localization by indirect immunofluorescence. Planta 167 : 444–451. Smith MT. 1991. Studies on the anhydrous fixation of dry seeds of lettuce (Lactuca stia L.). New Phytologist 119 : 575–584. Stahl B, Klabunde T, Witzel H, Krebs B, Steup M, Karas M, Hillenkamp F. 1994. The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of red kidney bean. Determination of the structure by a combination of matrix-assisted laser desorption}ionization mass spectrometry and selective enzymic degradation. European Journal of Biochemistry 220 : 321–330. Stra$ ter N, Klabunde T, Tucker P, Witzel H, Krebs B. 1995. Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site. Science 268 : 1489–1492. Stra$ ter N, Fro$ hlich R, Schiemann A, Krebs B, Ko$ rner M, Suerbaum H, Witzel H. 1992. Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean. Journal of Molecular Biology 224 : 511–513. Suerbaum H, Ko$ rner M, Witzel H, Althaus E, Mosel B-D, Mu$ llerWarmuth W. 1993. Zn-exchange and Mo$ ssbauer studies on the [Fe–Fe] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans. European Journal of Biochemistry 214 : 313–321. Towbin H, Staehlin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proceedings of the National Academy of Sciences of the United States 76 : 4350–4354. Ullah AHJ, Gibson DM. 1988. Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds. Archies of Biochemistry and Biophysics 260 : 514–520. Vincent JB, Crowder MW, Averill BA. 1992. Hydrolysis of phosphate monoesters : a biological problem with multiple chemical solutions. Trends in Biochemical Sciences 17 : 105–110. Vincent JB, Olivier-Lilley GL, Averill BA. 1990. Proteins containing oxo-bridged dinuclear iron centers : a bioinorganic perspective. Chemical Reiews 90 : 1447–1467. Weiler EW. 1986. Plant hormone immunoassays based on monoclonal and polyclonal antibodies. In : Linskens HF, Jackson JF, eds. Immunology in plant sciences. Berlin : Springer-Verlag, 1–17.
Localization of the Fe(III)-Zn(II) Purple Acid Phosphatase in Dry Kidney Beans Using Immunofluorescence and Immunogold Electron Microscopy M. G R O TE*, C. S C H W E R D T F E G ER†, R. W I E R M A NN†‡, R. R E I C H E L T* and H. W I T Z EL§ * Institut fuX r Medizinische Physik und Biophysik, † Institut fuX r Botanik and § Institut fuX r Biochemie, WestfaX lische Wilhelms-UniersitaX t, MuX nster, Germany Received : 10 March 1998
Returned for revision : 26 March 1998
Accepted : 30 April 1998
Localization of purple acid phosphatase (PAP) from the seeds of kidney beans, Phaseolus ulgaris (L.), was performed using light and transmission electron microscopy. After rehydration and aqueous fixation, cryo-sections of bean cotyledon tissue showed a bright immunofluorescent signal in the cytoplasm of cells whereas cell walls and reserve materials (starch, protein bodies) remained unstained. In ultrathin sections of dry cotyledon tissue anhydrously fixed in acrolein vapour and embedded in Lowicryl resin, PAP mapped exclusively to ribosome-rich areas of the cytoplasm. In view of these results, we propose that kidney bean PAP might possibly be engaged in mechanisms involved in the triggering of seed dormancy. # 1998 Annals of Botany Company Key words : Phaseolus ulgaris (L.), kidney bean, purple acid phosphatase, immunofluorescence microscopy, immunogold electron microscopy, anhydrous vapour fixation, acrolein.
INTRODUCTION Acid phosphatases are ubiquitously distributed in plants, and occur in numerous tissues. Several classes of acid phosphatases from plant sources have been reported (Fujumoto, Nakagawa and Ohara, 1977 ; Beck et al., 1986 ; Ullah and Gibson, 1988 ; LeBansky, McKnight and Griffing, 1992 ; Ferte et al., 1993 ; Cashikar, Kumaresan and Rao, 1997 ; for review see Vincent, Crowder and Averill, 1992 ; Duff, Sarath and Plaxton, 1994). An intensely studied plant enzyme is purple acid phosphatase (PAP) from the dry seeds of kidney beans (Phaseolus ulgaris L.). Kidney bean (kb)PAP was purified to homogeneity and biochemically and biophysically characterized in detail (Beck et al., 1986 ; Ullah and Gibson, 1988 ; Suerbaum et al., 1993 ; Klabunde et al., 1994, 1995 ; Stahl et al., 1994 ; Stra$ ter et al., 1995). The enzyme was also sequenced (Klabunde et al., 1994) and crystallized (Stra$ ter et al., 1992). PAPs contain a dimetal centre in the active site (Doi, Antanaitis and Aisen, 1988 ; Vincent, Olivier-Lilley and Averill, 1990). PAP from kidney beans is a homodimeric Fe(III)-Zn(II) metalloenzyme with a molecular mass of 111 kD (Beck et al., 1986, 1988 a, b). By X-ray structure analysis the ligands for the two metal ions could be elucidated (Stra$ ter et al., 1995 ; Klabunde et al., 1996). Kidney bean PAP catalyses the hydrolysis of activated phosphoric acid esters and anhydrides like ATP at a pH range from 4 to 7. Disregarding the first 122 amino acids of kidney bean PAP N-terminus, its sequence shows less than 20 % similarity to uteroferrin, a purple acid phosphatase of mammalian origin (porcine uterus). In this connection it is ‡ For correspondence, E-mail Wierman!uni-muenster.de
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surprising that the ligands of the two metal ions are conserved so that the Zn(II) could be exchanged against Fe(II) without any loss of catalytic activity. Electron paramagnetic resonance (EPR) (Dietrich et al., 1991) and Mo$ ssbauer data (Suerbaum et al., 1993) of the Zn(II) exchanged Fe(III)-Fe(II) enzyme from kidney beans are nearly identical to those of the mammalian enzyme. The plant and the mammalian enzymes are possibly derived from a common ancestral enzyme. During evolution, PAP might have taken over different functions in plant and mammalian lines. Despite this wealth of structural information on kidney bean PAP, there are few data concerning the precise location and the physiological role of this enzyme. Klabunde et al. (1995) suggested an anti-oxidant function for the enzyme from kidney beans. Using enzyme cytochemistry by phosphatase activity staining, Cashikar et al. (1997) localized kbPAP in the cell walls of the peripheral cotyledon cells and argued that the enzyme might play a role in the nutrition of the developing embryo by mobilizing organic phosphate in the soil. In order to obtain more insight into the biological function of the enzyme, its precise cellular and subcellular location have to be established. Since most structural studies on kbPAP were performed on the enzyme extracted from the dry seed we chose to start here with our localization study. We used highly specific antibodies against kbPAP in combination with two independent immunomicroscopic techniques : immunofluorescence light microscopy for a histological survey, and immunogold electron microscopy for ultrastructural analysis. For the immunofluorescent approach, specimens were treated in the conventional way using aqueous fixation of rehydrated beans. However, for electron microscopy, dry plant tissue can be prepared in an anhydrous way, by, e.g. osmium vapour fixation (Hallam,
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
1976 ; Opik, 1980, 1985 ; Smith, 1991) thus preserving the dehydrated morphology of the cells. Since osmium fixation severely impairs antigenicity of most antigens (Griffiths, 1993), we modified this technique using acrolein vapour instead of osmium vapour as the fixative and combined this kind of anhydrous fixation with immunogold microscopy localizing water-soluble allergens in dry grass pollen (Grote et al., 1994).
Seeds of Phaseolus ulgaris (L.), the red kidney bean, were obtained from Mu$ ller’s Mu$ hle (Gelsenkirchen, Germany).
six washes (each lasting 5 min) with TBS containing 0±05 % (v}v) Tween 20, the strips were incubated with an antibodyalkaline phosphatase conjugate (goat anti-mouse IgG ; Atlanta, Heidelberg, Germany, No. S3721), diluted by the factor of 10% with blocking solution for 1 h. After six further washes [five with TBS containing 0±05 % (v}v) Tween 20 and one with TBS without Tween 20] the strips were incubated without agitation in substrate [66 ml 5 % (w}v) nitro-blue-tetrazolium-chloride, dissolved in 70 % (v}v) dimethylformamide, 33 ml 5 % (w}v) 5-bromo-4-chloro-3indolylphosphate dissolved in dimethylformamide and 9±9 ml 100 m Tris}HCl (pH 9±5) plus 100 m NaCl and 5 m MgCl ]. The reaction was terminated after 5–30 min # and strips were washed three times with water.
Enzyme preparation
Epifluorescence microscopy
Isolation and purification of the enzyme from dry seeds followed the procedure described by Klabunde et al. (1994) for determination of the amino acid sequence. 30 mg of pure enzyme can be obtained from 300 g bean meal after an extraction step, ethanol precipitation, ethanol fractionation, ammonium sulphate precipitation and finally an affinity chromatography step on Blue sepharose CL-4B.
Fixation and preparation of the freeze-sections. Dry seeds of Phaseolus ulgaris (L.) were soaked in water for 20 h before the red hull was removed and the cotyledons were cut into cubes of approx. 3–4 mm square. These tissue blocks were fixed in 4 % para-formaldehyde (w}v) in 50 m phosphate buffer, pH 7±2 for 4 h at room temperature. After fixation, the specimens were washed four times (each time for 10 min) in phosphate-buffered saline (PBS) buffer (10 m sodium phosphate buffer, 150 m NaCl), pH 7±4. Then they were embedded in freezing medium (Reichert and Jung, Heidelberg, Germany) and cut into 20 µm thick sections using a cryo-microtome (Frigocut 2700, Reichert and Jung, Heidelberg, Germany). Immunolabelling. In situ localization of purple acid phosphatase was performed following the method of Scha$ chtele and Steup (1986). Frozen-sections of bean cotyledon tissue were placed on slides. The thawed sections were washed three times (10 min each) with PBS buffer containing 0±5 % BSA, pH 7±4 at ambient temperature then incubated with the following solutions : (1) anti-purple phosphatase antibody, diluted 1 : 1600 in PBS, 1 h ; (2) washing in PBS, three¬10 min each ; (3) goat anti-mouse IgG-FITC (fluorescein isothiocyanate) conjugate diluted 1 : 125 in PBS, 1 h ; (4) washing in PBS, three¬10 min. Control sections were : (1) incubated with mouse preimmune antibodies instead of the specific antibodies ; and (2) the primary antibody was omitted and the sections incubated with the IgG-FITC conjugate alone. In situ localization by epifluorescence microscopy. After immunolabelling, sections were analysed by epifluorescence microscopy using a Leitz Diaplan fluorescence microscope (Leitz, Wetzlar, Germany) with filter blocks L3 and GR. Epifluorescence micrographs were recorded on Fujichrome 400 film.
MATERIALS AND METHODS Plant material
Preparation of crude extracts for antibody testing All steps of extraction were performed between 0–4 °C. 3 g of cotyledon tissue were homogenized in 5 ml TBS (50 m Tris}HCl, 150 m NaCl, 1 m MgCl , pH 7±8). The # extract was filtered through four layers of nylon gauze and subsequently centrifuged in a Sorvall SS-34 rotor at 10 000 rpm for 10 min. The supernatant was mixed with sample buffer and used for electrophoresis. Antibody preparation Four-week-old female Balb c mice were immunized according to Weiler (1986). Each injection contained 20 mg of purified kidney bean purple acid phosphatase in incomplete Freund’s adjuvant. Primary injections were given intraperitoneally at day 1 and day 7. Further booster injections were given every 4 weeks. Preimmune serum was collected before first priming. 100–200 ml serum samples were collected 1 and 2 weeks after each booster injection by tail vein bleeding and pooled for experimental use. SDS-PAGE and immunoblotting SDS-PAGE was performed according to Laemmli (1970). The sample was incubated at 95 °C for 5 min and 50 mg protein was applied per cm gel. It was run at 15 mA in stacking gel and 20 mA in separation gel at room temperature. After transfer of the separated proteins onto nitrocellulose (Towbin, Staehlin and Gordon, 1979), the blot was cut into strips for probing with antibodies. First, the strips were incubated for 1 h with TBS containing 0±05 % (v}v) Tween 20 and 3 % (w}v) powdered milk (blocking solution) under constant agitation. The solution was then substituted by serum diluted by the factor of 5¬10$ with blocking solution and incubated for 1 h. After
Electron microscopy Anhydrous fixation. To gain information from the original dehydrated state of the specimen, fixation and preparation techniques for electron microscopy were kept strictly anhydrous. Since preliminary experiments showed that anhydrous fixation in paraformaldehyde vapour (a mild form of fixation preserving antigenicty of most antigens ; Griffith, 1993) rendered unsectionable specimens, fixation
Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans was performed in acrolein vapour (Grote et al., 1994). After removing the seed shell, cubes of approx. 1 mm$ were excised with a sharp razor blade from the peripheral part of the cotyledons and placed on cover slips. The cover slips were put into a desiccator together with a Petri dish filled with acrolein liquid (Riedel-de-Haen, Seelze, Germany). The desiccator was tightly sealed and placed into a fume hood at ambient temperature. Samples stayed in acrolein vapour for 13 d since preliminary experiments showed that shorter periods of fixation tended to result in unsectionable specimens. Dehydration and embedding. After vapour fixation, samples were completely dehydrated in dimethoxypropane (DMP) and absolute ethanol for 4 h each and then transferred successively into four ethanol : Lowicryl K4M mixtures (2 : 1, overnight ; 1 : 1, 8 h ; 1 : 2, overnight ; 1 : 5, 8 h). Afterwards, they were placed into pure Lowicryl resin where they remained for 48 h with two changes of the medium at room temperature. Polymerization was carried out at ®35 °C after the samples had been placed in fresh resin and slowly cooled over a period of 4 h to ®35 °C before UV irradiation was started. Sectioning. Ultrathin sections were cut on a Reichert OM U3 ultramicrotome and placed on nickel grids for immunolabelling procedures. Immunolabelling. Ultrathin sections on nickel grids (room temperature, moist chamber, PBS buffer at pH 7±4, TrisBSA buffer at pH 8±2) were put on droplets of the following solutions : (1) 5 % BSA in PBS, 15 min ; (2) mouse antipurple acid phosphatase antibody, diluted 1 : 2000 in PBS, 2 h ; (3) PBS, two¬5 min and Tris-BSA, 5 min ; (4) goat anti-mouse IgG coupled to 10 nm colloidal gold particles (GAM IgG 10) diluted 1 : 10 in Tris-BSA, 1 h ; (5) Tris-BSA, 5 min and distilled water, two¬5 min. Controls. Controls were performed (1) by replacing the primary specific antibody with mouse preimmune antibodies and (2) by omitting the primary specific antibody and incubating the sections with the gold-conjugate alone. Staining. After immunoabelling, sections were stained for 6 min in uranyl acetate and for 10 sec in lead citrate. Electron microscopy and documentation. After immunocytochemistry and staining, sections were analysed in a Hitachi H500 transmission electron microscope operated at 75 kV. Photographs were taken using Agfa-Gevaert 23D56 film. RESULTS Specificity of antibodies When crude extracts of Phaseolus ulgaris (L.) seeds were blotted onto nitrocellulose and tested with the antibodies, a strong specificity to purple acid phosphatase was shown (Fig. 1). For this reason they were used for immunolabelling experiments.
Immunofluorescence localization of PAP Whereas cell walls are devoid of fluorescent staining, a bright fluorescent signal is given by the cytoplasm in all cells
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F. 1. Immunoblot showing the reactivity of crude extract of dry seeds of Phaseolus ulgaris (L.) with mouse antibodies directed against purple acid phosphatase. Lane a, Crude extract stained with ponceau S ; lane b, crude extract stained with preimmune serum ; lane c, crude extract stained with specific immune serum. (The faint staining below the 52 kD PAP main band most probably results from the fact that the enzyme may occur with variable degrees of glycosylation. This would lead to an electrophoretical separation of the differently glycosylated molecules.)
of the cotyledons with the exception of the vascular tissue (Fig. 2 A and B). However, starch grains and numerous small particles of 2±2–2±5 µm diameter (protein bodies) do not show immunofluorescence. After treating the sections with pre-immune serum, as well as with IgG-FITC conjugate alone, a negligible degree of non-specific background fluorescence can be observed (Fig. 2 C–E). Similar results concerning the localization of purple acid phosphatase were obtained by laser scanning microscopy (data not shown).
Immunogold localization of PAP After incubating ultrathin sections of bean cotyledon tissue with anti-PAP antibodies and gold-conjugated secondary antibodies (Fig. 3 A), gold particles bind specifically to the cytoplasm of the cells but not the cell walls. Within the cytoplasm, protein bodies, amyloplasts (not shown in Fig. 3 A) and mitochondria show almost no decoration with the gold label. However, numerous gold particles are found associated with accumulations of ribosomes, which fill the spaces between the various cell organelles and inclusions of reserve materials throughout the cell. Control sections incubated with the preimmune antibodies revealed a low degree of non-specific staining (Fig. 3 B). Sections incubated with the gold-conjugated secondary antibody alone were almost completely free from gold particles (micrograph not shown).
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
A
B
C
D
E
F. 2. Immunofluorescence localization of PAP in rehydrated seeds of Phaseolus ulgaris (L.). Cross-section of a cotyledon incubated with antipurple acid phosphatase antibodies (A, filter block L3 ; B, filter block GR), with preimmune serum (C, filter block L3 ; D, filter block GR) or with IgG-FITC conjugate alone (E, filter block GR). Bars ¯ 10 µm (A, C–E, ¬416 ; b, ¬1090).
DISCUSSION In the present study two independent microscopic techniques were used to localize PAP in kidney beans : immunofluorescence microscopy and immunogold electron microscopy. Immunofluorescence microscopy using cryo-
sections of rehydrated and aqueously fixed kidney beans revealed positive staining throughout the cytoplasmic matrix of the cotyledon cells. Neither cell walls nor reserve materials (starch, protein bodies) yielded specific fluorescence. This comparatively coarse screening was supplemented and specified by transmission electron mi-
Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
239
A
B F. 3. A, Immunoelectron microscopic localization of PAP in dry seeds of Phaseolus ulgaris (L.). Detail from cotyledon cell showing part of cell wall (W) and cytoplasm with mitochondria (M), accumulation of ribosomes (R) and protein bodies (P) after incubation with mouse antipurple acid phosphatase antibodies followed by 10 nm colloidal gold conjugated secondary antibodies. Gold particles predominantly map to ribosome-rich areas in the cytoplasm. Bar ¯ 0±2 µm. B, Immunoelectron microscopic control experiment. Detail of similar cell compartment as in A after incubation with mouse preimmune antibodies followed by 10 nm colloidal gold-conjugated secondary antibodies. Few non-specifically bound gold particles are observed. Bar ¯ 0±2 µm.
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Grote et al.—Localization of Purple Acid Phosphatase in Dry Kidney Beans
croscopy. Since conventionally fixed and embedded dry kidney beans—besides rendering almost unsectionable specimens—do not reflect the original dehydrated state of the cells, we used an alternative fixation technique, i.e. vapour fixation. In a modified form, using acrolein vapour instead of osmium vapour, this technique had been applied in our lab for the immunocytochemical detection of highly water-soluble allergens in grass pollen species (Grote et al., 1994). Applying this anhydrous preparation technique for the first time in the localization of kbPAP eliminated any suspicion of a possible rehydration of the cells which might have been accompanied by alterations of cellular morphology including possible dislocation of the enzyme. Thus the truly desiccated state of the cells was not broken. Immunogold electron microscopy of anhydrously prepared kidney beans mapped PAP to ribosome-rich areas within the cytoplasm of the cotyledon cells. Similar to our light microscopic findings, no labelling was found in the cell walls, starch grains or protein bodies. Though gold particles seem to be located freely in the cytosol, we cannot exclude the possibility that they are confined to membrane-bounded cellular compartments or vesicles. Electron microscopy of dry bean cells, as is true of dehydrated tissues in general, presents a special and somewhat puzzling aspect of cellular morphology if compared with the more ‘ normal ’ look of hydrated cells. Thus, the cytoplasm shows many vacuolar spaces and coils of membranes which are not always easy to interpret and assign to conventional cellular compartments and organelles. For the two immunohistochemical approaches used, pretreatment of the plant material was not identical. For light microscopic experiments, beans were rehydrated and aqueously fixed, whereas electron microscopy was performed on dry tissue. Thus, strictly speaking, the results of the two techniques might not be considered comparable. However, both immunofluorescence and immunoelectron microscopy yielded labelling patterns which are fully compatible. Gross dislocations such as migration of PAP from the cytoplasm into the cell walls which would have been visible under the light microscope did not take place during the short initial rehydration period. This conclusion must be drawn from the results of electron microscopy which represent the labelling status of the original dry kidney bean cell. In the plant tissues studied to date (Giordani, 1978 ; Matile, 1978 ; Giordani et al., 1986 ; Ferte et al., 1993 ; Duff et al., 1994) acid phosphatases were generally localized in a number of different cell compartments such as vacuoles, cytosol and cell walls. Extracellular phosphatases appear to be ubiquitous in roots and plant cell cultures (Ferte et al., 1993 ; Duff et al., 1994). In laticifers acid phosphatases were located in the lumen of provacuoles and vacuolar apparatus (Giordani et al., 1986). The authors suggested that these enzymes are involved in the turnover of cytoplasmic components (cf. Matile, 1978). Concerning the localization of kidney bean PAP, a recent report (Cashikar et al., 1997) found the enzyme exclusively in the walls of the peripheral cotyledon cells. These authors applied a histochemical procedure which uses ATP as substrate for PAP : free phosphate released on PAP catalysis was precipitated as
insoluble lead phosphate. This technique, however, does not distinguish between different enzymes ; it would simply indicate any ATP-hydrolysing system. From their cytochemical observations, Cashikar et al. (1997) hypothesized that kidney bean PAP is involved in the mobilization of organic phosphates in the soil for the developing embryo. In view of the present immuno-electron microscopic results which show PAP to be associated with intracellular areas rich in ribosomes their hypothesis does not appear plausible. PAPs from human macrophages seem to be involved in killing microbes and the degradation of bone by production of free radicals. This supports the hypothesis that animal PAPs are involved in the formation of species of active oxygen (Suerbaum et al., 1993 ; Hayman and Cox, 1994 ; Klabunde et al., 1995). PAP from kidney beans, however, does not seem to function in such a manner because its Fe(III)-Zn(II) centre cannot form free oxygen radicals. Moreover, its cytosolic localization is not compatible with such a function. Thus, at present, we still cannot define the exact biological function of PAP. Its localization pattern in ribosome-rich areas of the cytosol suggests that the enzyme might be involved in metabolic}catabolic processes whose nature, however, we do not yet understand. Since morphological and cytochemical investigations of the dry seed required specialized techniques, we felt it worthwhile to present our new information on the localization of PAP in dry kidney beans in a separate paper. However, it will be necessary to extend these studies by following the expression of this enzyme in various stages of seed development and germination. The origin and fate of PAP in kidney beans could be followed more closely thus providing additional clues to its biological function. A C K N O W L E D G E M E N TS The work was supported by the Deutsche Forschungsgemeinschaft (to H. W.). The authors appreciate the skilful technical assistance of Mr U. Malkus and the expert photography of Mrs G. Kiefermann. We are very grateful to Professor M. Steup (Institut fu$ r Biochmie und Molekulare Physiologie, Potsdam), and Dr B. Greve (Institut fu$ r Strahlenbiologie, Mu$ nster) for the generous gift of antisera against purple acid phosphatase from kidney beans and the performance of the immunoblot experiment. The research described in this paper was initiated by Prof. Dr. H. Witzel, who made fundamental contributions to the biochemistry and biophysics of purple acid phosphatases. He died on 1 Sept. 1996. LITERATURE CITED Beck JL, McArthur MJ, de Jersey J, Zerner B. 1988 a. Derivatives of the purple phosphatase from red kidney bean : replacement of zinc with other divalent metal ions. Inorganica et Chimica Acta 153 : 39–44. Beck JL, de Jersey J, Zerner B, Hendrich MP, Debrunner PG. 1988 b. Properties of the Fe(II)-Fe(III) derivative of red kidney bean purple phosphatase. Evidence for a binuclear Zn–Fe center in the native enzyme. Journal of the American Chemical Society 110 : 3317–3318.
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