[16] Solubilization of functional membrane proteins

Hjelmeland, Leonard M.; Chrambach, Andreas

doi:10.1016/s0076-6879(84)04097-0

Cited by 261 publications

(136 citation statements)

References 16 publications

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“…The solubilized protein was completely reduced by ascorbate, had a rather high pI value, and eluted from a Superdex 200 gel-filtration column as a 130 kDa detergent-protein complex. This is in agreement with the apparent molecular mass of about 90 kDa of a Triton X-100 micelle (Hjelmeland & Chrambach 1984) and the molecular mass calculated for the cyt. b 561 from chromaffin granule of 27-29 kDa (Perin et al 1988;Annaert 1993).…”

Section: Hemessupporting

confidence: 91%

Redox enzymes in the plant plasma membrane and their possible roles

Bérczi

Møller

2000

Plant Cell & Environment

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Section: Hemessupporting

confidence: 91%

Redox enzymes in the plant plasma membrane and their possible roles

Bérczi

Møller

2000

Plant Cell & Environment

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“…Calcium uptake was rapid in the presence of a H' gradient but only slight in its absence showing that a H' gradient is required for calcium uptake. Calcium transport increased twofold when the pHo., was increased from 7 (Fig. 4D) to 8 ( Fig.…”

Section: Resultsmentioning

confidence: 97%

“…1). All three detergents solubilized proteins maximally at concentrations well above their critical micelle concentrations; however, octylglucoside and cholate were chosen for further study as they have much higher critical micelle concentrations (25 and 8 mm, respectively) when compared to 0.3 mm for Triton (7). Both detergents have been successfully used in solubilization and reconstitution of calcium transport proteins from animal and bacterial cell membranes (2,3,6,11,13,16).…”

Section: Resultsmentioning

confidence: 99%

Solubilization and Reconstitution of the Oat Root Vacuolar H⁺/Ca²⁺ Exchanger

Schumaker¹,

Sze²

1990

Plant Physiol.

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Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H+/Ca2+ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ApH] dependent). The first step toward purification and identification of the H+/Ca2+ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H+/Ca2+ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ApH (acid inside) was generated in the proteoliposomes with an NH4CI gradient (NH4+in>> NH4+,A) as determined by methylamine uptake. Fundamental properties of ApH dependent calcium uptake such as the Km for calcium (-15 micromolar) and the sensitivity to inhibitors such as N,N'-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H+/Ca2+ antiporter has been reconstituted in active form.inhibited by low levels of DCCD,3 ruthenium red, and lanthanum (15).To understand the function of the transporter at the molecular level, it will be necessary to isolate and purify the protein responsible for H+/Ca2" exchange. A first step toward this goal is to develop an assay to measure transport activity after solubilization and during subsequent purification. This study describes the solubilization of the oat root vacuolar H+/Ca2" exchanger and its reconstitution into liposomes. The reconstituted calcium transport activity has similar affinity for calcium and displays the same inhibitor sensitivities as previously found for the native H+/Ca2" transporter of the oat root vacuole. To our knowledge, this is the first report of the reconstitution of a functionally active H+-coupled transport system from plants. MATERIALS AND METHODS Plant MaterialOat seeds (Avena sativa L. var Lang) were germinated in the dark over an aerated solution of 0.5 mM CaSO4. Roots were harvested after 4 d. Preparation of Membrane VesiclesVesicles were prepared by a modification of an earlier procedure (15). All procedures were conducted at 4°C. Briefly, oat roots (20-60 g) were homogenized by mortar and pestle in a medium containing 250 mM sorbitol, 3 mM EGTA, 25 mM Hepes-BTP (pH 7.4), 1 mM DTT, and 0.2% BSA at a medium-to-tissue ratio of 1.5 mL/g fresh weight. After filtration through cheesecloth, the debris was rehomogenized in 1 mL/g of the original tissue weight, washed in 0.5 mL/g and filtered. The homogenate was centrifuged for 15 min at 13,000g, and the supernatant was centrifuged for 30 min at 60,000g (Beckman SW 28 rotor, rmax). The resulting pellet (crude microsomal pellet) was resuspended in 250 mM sorbitol, 2.5 mm Hepes-BTP (pH 7.2), and 1 mM DTT (resuspension buffer). The suspension (6 mL) was layered over a 6% dextran (w/w) cushion (10 mL) prepared in resuspension buffer. After centrifugation for 2 h at 70,000g (SW 28. 1, rmax), a turbid band a...

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“…This is most effectively accomplished by using amphiphilic detergents and the process is known as solubilization (Banerjee, 1999;Hjelmeland and Chrambach, 1984;Jones et al, 1987). Effective solubilization and purification of membrane receptors in a functionally active form represents an important step in understanding structure-function relationship and pharmacological characterization of a specific receptor.…”

Section: Introductionmentioning

confidence: 99%

Solubilization of Serotonin_1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells

2004

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SUMMARY1. The serotonin 1A (5-HT 1A ) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions.2. We report here, for the first time, the solubilization of 5-HT 1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT 1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration.3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT 1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.

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[16] Solubilization of functional membrane proteins

Cited by 261 publications

References 16 publications

Redox enzymes in the plant plasma membrane and their possible roles

Redox enzymes in the plant plasma membrane and their possible roles

Solubilization and Reconstitution of the Oat Root Vacuolar H⁺/Ca²⁺ Exchanger

Solubilization of Serotonin_1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells

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[16] Solubilization of functional membrane proteins

Cited by 261 publications

References 16 publications

Redox enzymes in the plant plasma membrane and their possible roles

Redox enzymes in the plant plasma membrane and their possible roles

Solubilization and Reconstitution of the Oat Root Vacuolar H+/Ca2+ Exchanger

Solubilization of Serotonin1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells

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Product

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Solubilization and Reconstitution of the Oat Root Vacuolar H⁺/Ca²⁺ Exchanger

Solubilization of Serotonin_1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells