Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

Crespi, Pierre; Perroud, Pierre‐François; Greppin, Hubert

doi:10.1007/bf00262642

Cited by 11 publications

(11 citation statements)

References 42 publications

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“…As an important preparative purification technique [1][2][3][4][5], freeflow electrophoresis (FFE) has been used for separation of small ions [6,7], DNA [8,9], peptides [10,11], proteins [12][13][14], and membrane vesicles [15,16] as well as cells [17][18][19][20]. Due to its complete liquid-phase electrophoresis, FFE has numerous advantages, such as being with quite gentle and controllable experiment conditions, relatively high-yield preparation, well protection of sample activity, and wholly continuous sepa-ration mode in contrast to a batch one in chromatography [21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

et al. 2012

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Interval free-flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free-flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10⁻³). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two-model antibiotics (herein pyoluteorin and phenazine-1-carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

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Section: Introductionmentioning

confidence: 99%

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

et al. 2012

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“…Rab GTPases have a major function in intracellular protein traffic between organelle compartments and the plasma membrane, as these proteins are also involved in secretion and endocytosis (Bischoff et al 1999). These proteins appear to be constitutive as they are present in control samples and GTP-binding proteins of similar mass (17-35 kDa) have been found in many other plant membrane preparations (see Ma 1994;Bischoff et al 1999) such as S. oleracea plasma membranes (Crespi et al 1996) or tonoplast (Perroud et al 1997) and microsomes of Pisum sativum (Novikova et al 1997). Interestingly, two bands at $25 and $28 kDa in plasma membrane fractions extracted from NodNGR[S] pretreated roots have a [ 35 S]GTPcS signal 1.5-fold and 1.3-fold, respectively, higher than the con-trol plasma membrane fractions (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…[ 35 S]GTPcS overlays [ 35 S]GTPcS overlays were conducted essentially as in Crespi et al (1996). Proteins were separated by SDS-PAGE (12% gels) and electrotransferred to nitrocellulose membranes that were dried overnight before saturation in overlay buffer (50 mM Tris pH 7.4, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA and 0.05% Tween 20) with rocking for 1 h. The membranes were incubated for 2 h in 10 ml of overlay buffer containing 1.7 nM [ 35 S]GTPcS and 100 lM ATP, then washed three times for 30 min each in overlay buffer and left to dry overnight.…”

Section: Preparation Of Membrane Fractionsmentioning

confidence: 99%

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp

Kelly

Irving

2003

Planta

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Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]-(from Rhizobium sp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata (L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [ 35 S]GTPcS, yielding a two-site displacement curve with displacement constants (K i ) of 0.58 lM and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K i of 4.4 and 29 lM, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N-N'-N''-N'''-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPcS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein ($55 kDa) recognised by Ga common antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

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“…While in the MS‐FFE, the gravity‐induced pressure is the difference between the liquid level in the background buffer reservoir and that of outlets positioned at the 16‐channel pump. Because of these reasons, the mode of sample injection herein is also different from those in the LS‐FFE 2, 3, 7–9, 12, 13, 20–22, 24–28 and the developed LS‐FEE 36–38 as discussed below.…”

Section: Design Of Ms‐ffementioning

confidence: 99%

“…proteins 13–19 and DNA 20), and sub‐cellular structures (e.g. membranes 21 and mitochondria 22, 23). Furthermore, LS‐FFE has been widely used for the isolation of human blood cells 24, bone marrow cells 25, kidney cells 26, tumor cells 27 and bacteria 7.…”

Section: Introductionmentioning

confidence: 99%

Mid‐scale free‐flow electrophoresis with gravity‐induced uniform flow of background buffer in chamber for the separation of cells and proteins

Dong

Shao

Yin

et al. 2011

J of Separation Science

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A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

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Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

Cited by 11 publications

References 42 publications

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp

Mid‐scale free‐flow electrophoresis with gravity‐induced uniform flow of background buffer in chamber for the separation of cells and proteins

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