Elution of acid phosphatase from sycamore cell walls

Plant Science, 40 (1985) 35--41 Elsevier Scientific Publishers Ireland Ltd.

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E L U T I O N O F ACID PHOSPHATASE F R O M SYCAMORE CELL WALLS

MARTINE CRASNIER* and ROGER OIORDANI**

Centre de Biochimie et de Biologic Moldculaire du CNRS, 31, Chemin Joseph Aiguier, P.B. 71, 13402 MarseiUe Cedex 9 (France) (Received December 21st, 1984) (Revision received May 15th, 1985) (Accepted May 15th, 1985) In isolated cell wall fragments of cultured sycamore cells, acid phosphatase activity was preferentially located on one side. This side has been identified as the external side of the cell wall. About 60% of acid phosphatase activity could be solubilized from cell wall fragments. Electron micrographs showed that the majority of the solubilized enzyme came from the external area of the wall.

Key words: cell wall; acid phosphatase; cytochemical localization

Introduction

Plant cell walls contain enzymes with hydrolytic activities, some of which are implicated in cell extension. Work with cell suspensions showed that several of these enzymes, especially acid phosphatase [1] and peroxidase [2,3], were located on t h e external area of the wall which is particularly rich in pectic substances [3]. Enzymes b o u n d to the cell wall can be released b y washing with solutions of various ionic strengths. The amount of released e n z y m e depends u p o n the t y p e of cell used and the specific enzyme studied [4,5]. Another w a y to extract cell wall proteins is b y the use of commercial enzymes which can degrade cell walls in vitro [6]. Acid phosphatase activity (EC 3.1.3.2) has been detected [7,8] in cell walls of *To whom correspondence should be sent at: Department of Biochemistry, The University of Birmingham, P.O. Box 363, Birmingham B15 2TT U.K. **Present address: Biologic de la Differenciation Cellulaire, L.A. CNRS 179. Facult~ des Sciences de Luminy, Case 901 13288 Marseille, Cedex 9, France. Abbreviations: PNP, p-nitrophen01; PNPP, p-nitrophenylphosphate.

cultured sycamore ceils. Cytochemical tests have localized the enzyme on t h e external side of the cell wall [9]. A t t e m p t s to solubilize the acid phosphatase o f cultured sycam o r e cell fragments failed to solubilize completely the enzyme. Thus treatment of cell wall fragments with 1 M NaC1 solubilized only a b o u t 60% of the acid phosphatase [ 1 0 ] . It could be argued that covalent binding could account for partial elution of the enzyme as well as the embedding of the enzyme in the cell wall. Nevertheless, the fact that the acid phosphatase cannot be completely solubilized suggests an heterogeneous distribution in the cell wall fragments. Interesting questions that naturally arise are: where is t h e enzyme localized which is susceptible to solubilization and what is the distribution of the enzyme that remains bound? The aim of this paper is to answer these questions b y using cytochemical tests. Materials and m e t h o d s Cell cultures Sycamore cells were cultured in liquid medium under sterile conditions. The cell strain used has been derived from secondary c a m b i u m initially cultured b y Jeffs and

0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltcl. Printed and. Published in Ireland

36 N o r t h c o t e [ 1 1 ] . The composition of the medium was that of Lamport [12] as modified b y Lescure [13]. Erlenmeyer flasks on rotating shakers (50 rev./min) were illuminated b y continuous light (600 lux). Cells used in this study were taken at the end of the exponential growth phase.

Cell wall preparations Isolated cell walls were prepared according to the procedure already reported [ 1 0 ] . Cell disruption was achieved b y a French press under a pressure of 1000 kg/cm 2 in the presence of 0.4 M sucrose. The resulting suspension was centrifuged for 20 min at 1088 X g. The pellet was resuspended and centrifuged several times in sucrose solutions of increasing concentrations (0.6 and 1 M). The final pellet was carefully washed. In order to solubilize the acid phosphatase, cell wall fragments were treated three times with 1 M NaC1. Each treatment was performed at 4°C for 30 min.

Biochemical tests Acid phosphatase activity was followed b y measuring the l~ydrolysis of p-nitrophenylphosphate (PNPP). The standard incubation medium (3 ml) contained 0.05 M succinate buffer (pH 5.6), 0.66.10 -3 M PNPP and the enzyme sample (solubilized enzyme or cell wall fragments). After incubation at 30°C, the reaction was stopped b y 1 M NaOH and the absorbance read at 400 nm, When cell wall fragments were used as an enzyme sample, filtration of the reaction mixture was performed before absorbance reading. A unit of phosphatase activity was defined as the amount of p-nitrophenol (PNP) released ( m m o l . min -~ ) under standard assay conditions, Carbohydrate concentration was estimated b y the m e t h o d of Dubois et al. [ 1 4 ] .

Ultrastructural phatase

localization

of acid phos-

Specimens were pre-fixed in a mixture of 3% glutaraldehyde and 2.5% paraformaldehyde in a 0.1 M sodium phosphate buffer

(pH 7.3). Specimens were stained b y Gomori's m e t h o d [ 1 5 ] . Sections post-fixed with osmic acid were cut with a Porter Blum Model MT2 t y p e ultra-microtome, and stained fragments were viewed in a Philips EM 300 electron microscope at 60 KV. Cell wall enzyme activity was estimated b y counted lead precipitates. It was assumed that the collision risk b e t w e e n parietal enzyme and substrate (sodium ~-glycerophosphate) was the same in all space direction. The smallest lead precipitate was defined a~ the reaction p r o d u c t of one acid phosphatase activity unit. Results

In the initial steps of the preparation (steps a, b, c in Fig. 1), the cell wall fragments were accompanied b y nuclei and other cellular organelles. The elimination of the

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Fig. 1. Control o f purity o f the cell wall fragments. Cell wall fragments were prepared as described in Materials and methods. At each step o f purification, the absorhance at 280 nm (closed circles) and 260 nm (open circles) o f each washing solution was measured. In the final steps, carbohydrate concentration was also measured (A,90nm). a, b, c, d--i represent different steps o f purification as follows: a, cell disruption in presence o f 0.4 M sucrose; b, washing by 0.6 M sucrose; c, washing by 1 M sucrose; d--i, washing by distilled water.

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Solubilizotion steps Fig. 2. Solubilization of acid phosphatase from the cell wall fragments. Solubilization of acid phosphatase by 1 M NaCI was performed in the presence of succinic acid 0.05 M (pH 5.5) (open symbols) or in presence of Tris--HCl (pH 8.5) (closed symbols). In each case, the absorbance at 2 8 0 nm (circles) o f each washing solution was measured. The phosphatase activity (triangles) was also measured in standard conditions at pH 5.6. j, k, l--p represent different steps o f solubilization as follows: j, first washing by I M NaCI; k and 1, second and third washing by 1 M NaCI; m--p, washings by distilled water.

contaminating organelles was followed by measuring the absorbances at 260 n m and 280 n m of the washing solutions (Fig. I). Immediately after the first washing by distilled water, there was no contaminant absorbance, indicating that the cell walls were relatively clean. The cell wall fragments obtained in that way were viewed in an electron microscope and judged to be free from membrane contamination. About 6 5 % of phosphatase activity of the cell wall fragments could be solubilized by three washings of 1 M NaCl at p H 5.5.35% of the enzyme activity was eliminated during the first washing and only 7--10% during the third one. More washings did not solubilize significant amounts of enzyme (Fig. 2). The amount of eluted and non-eluted activity was similar to the total activity of the initial cell wall preparation, therefore elution treatment did not result in enzyme inactivation. The enzyme remaining bound could not be solubilized by further treatment with higher concentration of salt (5 M) or by the use of pectinase and cellulasemixtures.

Fig. 3. Acid phosphatase distribution in walls of intact cells examined by Gomori's method. Bars represent 0.25 ~m. Orig. mag. X 37 000.

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Fig. 4. Acid phosphatase distribution in isolated non-eluted cell wall fragments examined by Gomori's method. A, Heterogeneous distribution o f acid phosphatase. Orig. mag, X 42 000; B, Estimation o f the phosphatase activity by counting the lead precipitates in the enclosed area. The concentration of the granules was 3500 g • prn -2 in the external area and 440 g - u m -~ in the internal area. Orig. mag, X 37 000. C, Control incubated without substrate. Orig. mag. X32 000. Bars represent 0.25 ~m.

39

Fig. 5. Acid phosphatase distribution in eluted cell wall fragments examined by Gomori's method. A, Homogeneous distribution of acid phosphatase. Orig. mag. ×26 000. B, Estimation of the phosphatase activity b~/ counting the lead precipitates in the enclosed area. The concentration of the granules was 300 g • ~rn -~. Orig. mag. X42 000. C, Control incubated with substrate in the presence of 0.01 M sodium fluoride. Orig. mag. × 32 000. Bars represent 0.25/~m.

40

It was known from previous studies that pH can influence t h e extraction yield of cell wall proteins [ 1 6 ] , pH 8.5 being more effective than pH 5.5 [ 1 7 ] . When t h e salt extraction was performed at pH 8.5, we observed that although more protein was eluted than at pH 5.5 in agreement with previous results, a considerable proportion o f the acid phosphatase was not solubilized and still remained b o u n d (Fig. 2). In whole cells stained with Gomori's m e t h o d , the phosphatase activity was principally located on the external side of t h e wall (Fig. 3) as has been previously described [9]. When isolated non-eluted cell walls were examined b y the same m e t h o d , the phosphatase activity was preferentially located on one side (Fig. 4A). It could be assumed that this area corresponds to the external side of the wall. However, the phosphatase activity of non-eluted cell walls was not restricted to this area. Some of the enzyme activity was also distributed in the internal area of the wall (Fig. 4A). The lead precipitates were counted in t h e enclosed area of Fig. 4B. The external staining represented 56% of the total activity and the corresponding area was 14% of the wall (Fig. 4B). In control preparations which were incubated without substrate no reaction p r o d u c t could be detected {Fig. 4C). With eluted cell wall, the enzyme did not appear concentrated in the external area. The distribution of the remaining enzyme was uniform (Fig. 5A). Counting of lead precipitates (enclosed area of Fig. 5B) showed a distribution of the enzyme in the wall similar to the one in the internal area of the non-eluted cell wall (300 g r a n u l e s - u m -2 and 440 granules. /am -2, respectively). Thus the majority of the eluted enzyme came from the external area of the wall which contained 56% of the total phosphatase activity of the wall. Controls incubated with substrate in presence of 0.01 M sodium fluoride, an inhibitor of acid phosphatase [ 9 ] , showed no reaction product (Fig. 5C).

Conclusion

We can deduce from our micrographs that the acid phosphatase which can be solubilized is located on the external side of the wall. As this enzyme can be eluted by raising the ionic strength, it is probably electrostatically b o u n d to the cell wall. The rest of the enzyme remains strongly b o u n d to the cell wall at high salt concentration; this could involve covalent bonding which has been described for other cell wall proteins [ 1 8 ] . Cross linking b y amino acids such as isodityrosine could also be responsible for the insolubility of plant cell wall proteins [ 1 9 ] . However, another possibility for the enzyme not being eluted is that it could be embedded in the wall. It has been shown that pores of about 10 A diameter exist in the cell wall [ 2 0 ] . It is possible that an enzyme as big as acid phosphatase (100 000 daltons [10] ) can be kept mechanically inside the wall depending on its localization. The amounts of eluted cell wall acid phosphatase were quite similar whether measured b y biochemical or cytochemical tests (65% and 56%, respectively). The fact that different substrates were used for the localization and biochemical measurements of acid phosphatase m a y not constitute a problem as b o t h results were expressed on a percentage basis. However, misleading results could be obtained if the solubilized enzyme and the unsolubilized one corresponded to different isoenzymes with different relative affinities for b o t h substrates. This appeared not to be the case as kinetic studies have shown that the solubilized enzyme and the b o u n d enzyme showed t h e same Kin-value for PNPP [ 2 1 ] . Furthermore, the purified enzyme showed no presence of isoenzymes [ 1 0 ] . During the exponential growth phase of sycamore cell culture, the medium in which cells were grown was very low in acid phosphatase activity. This activity was a b o u t 2% of the total phosphatase activity of the cell wall. Thus, the presence of acid phosphatase

41

in the wall was unlikely to be due to adsorption o f enzyme from the medium. In cultured cells, the secretion of acid phosphatase in the medium is increased b y a decrease of phosphate in the nutrient medium [ 2 2 ] . It could be argued that this enzyme comes from the external area of the wall.

Acknowledgements We are grateful to Dr. Maria Luz Cardenas for helpful discussion. We are indebted to Professor S.V. Perry for allowing us to use the facilities of the Department of Biochemistry, University of Birmingham, during the preparation of this manuscript.

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