Abstract
An effective method for the preparation of purified cell walls from mesophyll cells of Valerianella olitoria has been developed. Cells were isolated by a mechanical procedure only and crude cell walls were prepared from cell homogenates. Crude wall suspensions were fractionated in a discontinuous sucrose gradient and the wall fragments recovered were examined by scanning electron microscopy. An evaluation of the degree of purity and physiological integrity of the wall fragments showed that the material found at the 50–60% (w/w) interface consisted mostly of wall particles of high purity. Some characteristics of the purified walls are reported, especially the following enzyme activities: β-d-glucosidase (EC 3.2.1.21) and the β-d-glucanases, 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), 1,4-β-glucan cellobiohydrolase (EC 3.2.1.91), 1,3-β-glucan glucanohydrolase (EC 3.2.1.39), 1,3-β-glucan glucohydrolase (EC 3.2.1.58). The results provided evidence for the microlocalization of some hydrolases and indicated that enzymes extracted only with a high-salt-concentration buffer were confined to walls whereas the 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-solubilized enzymes could have multiple sites, e.g. walls and membranes of the endoplasmic reticulum.
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Abbreviations
- CMC:
-
carboxymethylcellulose
- Tris:
-
2-amino-2-(hydroxymethyl)-1,3-propanediol
- PM:
-
plasma membrane(s)
- ER:
-
endoplasmic reticulum
References
Bal, A.K., Verma, D.P.S., Byrne, H., Maclachlan, G.A. (1976) Subcellular localization of cellulases in auxin-treated pea. J. Cell Biol. 69, 97–105
Bernstein, E.O., Jones, C.B. (1969) Skin replication procedure for the scanning electron microscope. Science 166, 252–253
Bowles, D.J., Kauss, H. (1976) Characterization, enzymic and lectin properties of isolated membranes from Phaseolus aureus. Biochim. Biophys. Acta 443, 360–374
Chanzy, H., Henrissat, B. (1983) Electron microscopy study of the enzymic hydrolysis of Valonia cellulose. Carbohydrate Polymers 3, 161–173
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356
Edwards, G.E., Black, C.C. (1971) Isolation of mesophyll cells and bundle sheath cells from Digitaria sanguinalis (L). Scop. leaves and a scanning microscopy study of the internal leaf cell morphology. Plant Physiol. 47, 149–156
Elias, B.A., Givan, C.V. (1978) Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue. The case of citrate synthase in Pisum sativum L. Planta 142, 317–310
Fagerstam, L.G., Petterson, L.G. (1980) The 1,4-β-glucan cellobiohydrolase of Trichoderma reesei. FEBS Lett. 119, 97–100
Huber, D.J. (1981) Status on Zea coleoptile cell wall: β-d-glucanases and their native substrates. (Abstr.) Diss. Abstr. Int. B 41, 2, 2879
Huber, D.J., Nevins, D.J. (1981) Partial purification of endo-and exo-β-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis. Planta 151, 206–214
Koehler, D.E., Leonard, R.T., Vanderwoude, W.J., Linkins, A.E., Lewis, L.N. (1976) Association of latent cellulase activity with plasma membranes from kidney bean abscission zones. Plant Physiol. 58, 324–330
Kubicek, C.P. (1981) Release of carboxymethyl-cellulase and β-glucosidase from cell walls of Trichoderma reesi. Eur. J. Appl. Microbiol. Biotechnol 13, 226–229
Leonard, R.T., Hodges, T.K. (1973) Characteriszation of plasma membrane adenosine triphosphatase activity of oat roots. Plant Physiol. 48, 6–12
Leonard, R.T., Hotchkiss, C.W. (1976) Cation-stimulated adenosine triphosphatase activity and cation transport in corn roots. Plant Physiol. 58, 331–335
Leonard, R.T., Hotchkiss, C.W. (1978) Plasma-membranes associated adenosine triphosphatase activity of isolated cortex and stele from corn roots. Plant. Physiol. 61, 175–170
Lewis, L.N., Linkins, A., O'Sullivan, S., Reid, P.D. (1974) Two forms of cellulase in bean plants. In: Plant growth substances 1973, pp. 708–718. Hirokawa Publishing Co., Tokyo
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275
Maclachlan, G.A. (1977) Cellulose metabolism in growing cells. Trends in Biol. Sci. (TIBS) 226–228
Outlaw, W.H., Schimuck, C.L., Tolbert, N.E. (1976) Photosynthetic carbon metabolism in the palisade parenchyma and spongy parenchyma of Vicia faba (L.). Plant Physiol. 58, 186–189
Quail, P.H. (1979) Plant cell fractionation. Annu. Rev. Plant Physiol. 30, 425–84
Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19–23
Van der Wilden, W., Chrispells, H.J. (1983) Characterization of the isoenzymes of α-mannosidase located in the cell wall, protein bodies and endoplasmic reticulum of Phaseolus vulgaris cotyledons. Plant Physiol. 71, 82–87
Whatley, F.R., Arnon, D.I. (1963) Photosynthetic phosphorylation in plants. Methods Enzymol. 6, 308–313
Wheeler, K.P., Walker, J.A., Barber, D.M. (1975) Lipid requirement of the membrane sodium-plus-potassium iondependent adenosine triphosphatase system. Biochem. J. 146, 713–722
Wong, Y.S., Fincher, G.B., Maclachlan, G.a. (1977) Kinetic properties and substrate specificities of two cellulase from auxin-treated pea epicotyls. J. Biol. Chem. 252, 1402–1407
Wong, Y., Maclachlan, G.A. (1979) 1,3-d-glucanases from Pisum sativum seedlings. II. Substrate specificities and enzymic action patterns. Biochim. Biophys. Acta 571, 256–269
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Lienart, Y., Barnoud, F. β-D-glucan-hydrolase activities in pure cell-wall-enriched fractions from Valerianella olitoria cells. Planta 165, 68–75 (1985). https://doi.org/10.1007/BF00392213
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DOI: https://doi.org/10.1007/BF00392213