Summary
Preparative free-flow electrophoresis has been employed in combination with density gradient centrifugation to prepare fractions enriched in either tonoplast or plasma membrane from dark grown seedlings of cress (roots), zucchini (hypocotyls), soybean (hypocotyls) and maize (coleoptiles). A polyclonal antibody to the 72,000 Mr subunit to the maize tonoplast ATPase was used to identify the tonoplast fractions from the free-flow electrophoresic separations and to show the absence of tonoplast contamination in plasma membranes derived from the same homogenates. These findings confirm the identity of the tonoplast fraction based on the presence of the proton translocating ATPase determined previously from sucrose gradient fractionation and inhibitor studies to be a tonoplast marker. Using staining with phosphotungstic acid at low pH, the plasma membrane fractions obtained after free-flow electrophoresis were shown to be > 90% plasma membrane-derived with little or no cross-contamination of plasma membrane vesicles in the tonoplast-containing fractions.
Finally, the composition of the fractions was correlated with the characteristic morphologic appearance after filipin treatment and freeze-fracture. By means of morphometric analyses using this criterion, both the identity and the purity of the tonoplast and the plasma membrane fractions received further confirmation. Essentially homogeneous fractions were obtained by subjecting fractions already enriched by a centrifugation method to the final separation by free-flow electrophoresis.
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Abbreviations
- BSA:
-
bovine serum albumin
- DMSO:
-
dimethylsulfoxide
- EF:
-
extraplasmatic fracture face
- ELISA:
-
enzyme-linked immune-adsorbance assay
- FIL:
-
filipin-induced lesion
- IMP:
-
intramembraneous particles
- PF:
-
plasmatic fracture face
References
Auderset G, Sandelius AS, Penel C, Brightman A, Greppin H, Morré DJ (1986) Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction. Physiol Plant 68: 1–12
Bowles DJ, Quail PH, Morré DJ, Hartmann GC (1979) Use of markers in plant-cell fractionation. In: Reid E (ed) Plant organelles. Horwood, Chichester, pp 207–224
Bowman EJ, Mandela S, Taiz L, Bowman BJ (1986) Structural studies of the vacuolar membrane ATPase fromNeurospora crassa and comparison with the tonoplast membrane ATPase fromZea mays. Proc Natl Acad Sci USA 83: 48–52
Gooderham K (1984) Transfer techniques in protein blotting. In: Walker JM (ed) Methods of molecular biology, vol 1. Humana Press, Clifton, NJ, pp 165–17
Kjellbom P, Larssen C (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol Plant 64: 398–403
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–675
Mandala S, Taiz L (1985) Partial purification of a tonoplast ATPase from corn coleoptiles. Plant Physiol 78: 327–333
— — (1986) Characterization of the subunit structure of the maize tonoplast ATPase. J Biol Chem 261: 12850–12855
Marty F, Branton D (1980) Analytical characterization of beetroot vacuole membrane. J Cell Biol 87: 72–83
Morré DJ (1973) Isolation and purification of organelles and endomembrane components from rat liver. In: Chrispeels MJ (ed) Molecular techniques and approaches in developmental biology. Wiley, New York, pp 1–27
—, Brightman A, Sandelius AS (1987) Membrane fractions from plant cells. In: Findlay JBC, Evans WH (eds) Biological membranes-a practical approach. IRL Press, Oxford, pp 37–72
Morré DM, Morré DJ, Bowen S, Reutter W, Windel K (1988) Vitamin A excess alters membrane flow in rat liver. Eur J Cell Biol 46: 307–315
Navas P, Nowack DD, Morré DJ (1989) Isolation of highly purified plasma membranes from cultured cells and hepatomas by twophase partition and preparative free-flow electrophoresis. Cancer Res 49: 2147–2156
Penel C, Auderset C, Bernardini N, Castillo FJ, Greppin H, Morré DJ (1988) Compositional changes associated with plasma membrane thickening during floral induction in spinach. Physiol Plant 93: 134–146
Quail PH (1979) Plant cell fractionation. Annu Rev Plant Physiol 30: 425–484
Roland J-C, Lembi CA, Morré DJ (1972) Phosphotungstic acid — chromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Technol 47: 195–200
Sandelius AS, Penel C, Auderset G, Brightman A, Millard M, Morré DJ (1986) Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soy bean hypocotyls by free-flow electrophoresis. Plant Physiol 81: 177–185
Scherer GFE (1984) Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation. Planta 160: 348–356
—, Fischer G (1985) Separation of tonoplast and plasma membrane H+-ATPase from zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation. Protoplasma 129: 109–119
Severs NJ, Robenek H (1983) Detection of microdomains in biomembranes. An appraisal of recent developments in freeze-fracture cytochemistry. Biochim Biophys Acta 737: 373–408
Steele ME, Stout RG (1986) Isoelectric focussing of oat root membranes. Plant Physiol 82: 327–329
Sun I, Morré DJ, Carne FL, Safranski K, Croze EM (1984) Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicles and Golgi apparatus fractions of rat liver. Biochim Biophys Acta 797: 266–275
Sze H (1985) H+-translocating ATPase: advances using membrane vesicles. Annu Rev Plant Physiol 36: 175–208
vom Dorp B, Volkmann D, Scherer GFE (1986) Identification of tonoplast and plasma membrane in membrane fractions from garden cress (Lepidium sativum L.) with and without filipin treatment. Planta 168: 151–160
Yoshida S, Uemura M (1986) Lipid composition of plasma membranes and tonoplasts isolated from etiolated seedlings on mung bean (Vigna radiata L.). Plant Physiol 82: 807–812
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vom Dorp, B., Scherer, G.F.E., Canut, H. et al. Identification of tonoplast fractions resolved from plasma membrane by free-flow electrophoresis using filipin-labeling and antibody to the tonoplast ATPase. Protoplasma 156, 57–66 (1990). https://doi.org/10.1007/BF01666506
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DOI: https://doi.org/10.1007/BF01666506