Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Hahn, Michael G.; Lerner, David R.; Fitter, Mindy S.; Norman, P. M.; Lamb, Chris

doi:10.1007/bf00392292

Cited by 35 publications

(27 citation statements)

References 19 publications

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“…164B4 was used, immunostaining of the barley membrane proteins was much more intense for proteins in the PM fraction than in the TG or ER fractions. This is different from the results of Lamb et al (18,25), who reported that the antibody labeled two membrane peaks on a linear sucrose gradient of tobacco membranes.…”

Section: Discussioncontrasting

confidence: 99%

“…164B4, that was prepared against proteins from the surface of tobacco protoplasts (18,25) reacted with a number of PM proteins in the 95-to 200-kD range, but cross-reacted most strongly with two protein bands of 100-and 114-kD (Fig. 2C, lane 3).…”

Section: Resultsmentioning

confidence: 99%

“…A monoclonal antibody to a surface protein of tobacco protoplasts (AB No. 164B4) (18,25) was a gift from C. Lamb, Salk Institute, San Diego, CA.…”

Section: Methodsmentioning

confidence: 99%

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separation and Immunological Characterization of Membrane Fractions from Barley Roots

DuPont¹,

Tanaka²,

Hurkman³

1988

Plant Physiol.

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Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) For studies of the relationship between environmental stress and ion transport in barley roots we needed to obtain both PM2 and tonoplast membranes, capable of in vitro transport, from the same roots. In a previous paper we demonstrated the separation of the PM and tonoplast H+-ATPases on continuous sucrose gradients (7). Tonoplast vesicles were collected on a step gradient and the properties of the tonoplast H+-ATPase were characterized in some detail (7,12). However, it was not certain that the PM ATPase could be adequately separated from the tonoplast ATPase and obtained in sufficient quantities on a step gradient so that detailed studies of transport by the PM ATPase could also be performed.There are few studies where sufficient quantities of sealed, The choice of tissue used for membrane isolation may determine whether higher rates of transport by PM vesicles (29) or tonoplast vesicles (2, 5, 6) are observed. The method of preparing the membranes also influences the type of vesicle that is obtained. For example Giannini et al. (14) reported that one grinding medium gave predominantly sealed, right-side-out tonoplast vesicles while another method gave predominately sealed, inside-out PM vesicles.Two-phase partitioning of membranes with PEG and dextran resulted in a highly purified PM fraction. The procedure was particularly valuable for green tissue because it was possible to obtain PM without heavy contamination with chloroplast membranes. However, the two-phase procedure was used only to prepare PM and not tonoplast, and the procedure selected for right-side-out PM vesicles (4, 21) that were not suitable for measurements of ATP-driven transport. Good measurements of in vitro transport were obtained by using the purified, reconstituted PM ATPase (1, 28). However, for many studies it would be desirable to start with intact membrane vesicles, in which the ATPase, antiports, and symports function as an integrated system. Continuous sucrose or dextran gradients are commonly used to separate intact tonoplast, PM and other membrane vesicles, although the degree of purity of the membrane fractions obtained has been questioned, and the yield of sealed, inside-out PM vesicles is generally much lower than the yield of sealed, rightside out tonoplast vesicles (2,5,7,31).In this paper ER, tonoplast, and PM vesicles were collected from the same homogenate, under identical conditions. We utilized SDS-polyacrylamide gels, antibodies to membrane proteins, and immunoblots to demonstrate that the PM ATPase was separated from the tonoplast ATPase on sucrose step gradients. We used immunoblots to locate the ATPase subunit...

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Section: Discussioncontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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separation and Immunological Characterization of Membrane Fractions from Barley Roots

DuPont¹,

Tanaka²,

Hurkman³

1988

Plant Physiol.

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“…Studies of the temporal, spatial, and developmental dynamics of plant cell-wall glycoconjugates have lagged behind corresponding animal studies because of the more limited collection of monoclonal antibodies against cell-wall carbohydrate epitopes that is available and because of the difficulty in characterizing the epitopes recognized by the available antibodies. Previous studies of plant cell-wall dynamics have focused on cell surface glycoproteins, since arrays of monoclonal antibodies have been generated against two types of such complex glycoconjugates, the arabinogalactan-proteins (Anderson et al, 1984;Brewin et al, 1985;Norman et al, 1986;Villanueva et al, 1986;Hahn et al, 1987;Knox et al, 1989Knox et al, ,1991Pennell et al, 1989Pennell et al, ,1991Horsley et al, 1993;Puhlmann et al, 1994;Kreuger and Van Holst, 1995) and Hyp-rich glycoproteins (Smallwood et al, 1994(Smallwood et al, , 1995Knox et al, 1995). Immunohistochemical studies have documented subcellular (Herman and Lamb, 1992;Rae et al, 1992;Van Aelst and Van Went, 1992;Horsley et al, 1993;Sherrier and VandenBosch, 1994) and cell-type-specific (Knox et al, ,1991Stacey et al, 1990;Van Aelst and Van Went, 1992;Benfey et al, 1993;Schindler et al, 1995) distribution of arabinogalactan epitopes.…”

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Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

et al. 1996

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The plant cell wall is a dynamic structure that plays important roles i n growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present i n plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis fhaliana seedlings. An epitope i n the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls i n a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal a-fucosyl-containing epitope is present i n almost all of the cell walls in the root. An arabinosylated (1 +6)-P-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated i n a temporal, spatial, and developmental manner.The understanding of the role(s) of cell walls in plant biology has evolved considerably in recent years. Whereas the function of cell walls was initially viewed as primarily providing form and structure to plant cells, it has now become clear that the wall has a variety of other functions in plant growth and development (Roberts, 1990) and in the interactions of plants with their environment and other organisms (Hahn et al., 1989). It is therefore important to learn where the macromolecular components. are located within the walls of individual cells and within tissues and how the structures of the wall polymers change as a function of plant growth and development and during active defense against disease and environmental stress.Monoclonal antibodies have proven to be valuable tools for studies of the dynamics of cell surface glycoconjugates in animals (Hakomori, 1984; Feizi and Childs, 1987). Studies of the temporal, spatial, and developmental dynamics of plant cell-wall glycoconjugates have lagged behind corresponding animal studies because of the more limited collection of monoclonal antibodies against cell-wall carbohydrate epitopes that is available and because of the difficulty in characterizing the epitopes recognized by the available antibodies. Previous studies of plant cell-wall dynamics have focused on cell surface glycoproteins, since arrays of monoclonal antibodies have been generated against two types of such complex glycoconjugates, the arabinogalactan-proteins (Anderson et al., 1984;

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“…Specific antibodies targeted at differentially expressed antigens have proven useful in a number of laboratories for purification and immunohistochemical localization of plant target molecules (4,6,8,18).…”

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Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant

Lynes¹

1992

Plant Physiol.

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Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Cited by 35 publications

References 19 publications

separation and Immunological Characterization of Membrane Fractions from Barley Roots

separation and Immunological Characterization of Membrane Fractions from Barley Roots

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant

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