Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column

Krämer, Reinhard; Heberger, Claudia

doi:10.1016/0005-2736(86)90269-5

Cited by 111 publications

(28 citation statements)

References 14 publications

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“…The glutamine/amino acid transporter was reconstituted by removing the detergent with a hydrophobic chromatography column [16,30]. In this procedure, the mixed micelles containing detergent, protein and phospholipids were repeatedly passed through the same Amberlite XAD-4 column.…”

Section: Reconstitution Of the Glutamine/amino Acid Transporter Into mentioning

confidence: 99%

“…In this procedure, the mixed micelles containing detergent, protein and phospholipids were repeatedly passed through the same Amberlite XAD-4 column. The composition of the initial mixture used for reconstitution was: 25 µl of the solubilized protein (25-35 µg protein in 1.3% C 12 E 8 ), 75 µl of 10 % C 12 E 8 , 100 µl of 10% egg yolk phospholipids in the form of sonicated liposomes prepared as previously described [30], 30 mM L-glutamine (except where differently specified), 4 mM ATP, 20 mM Hepes/Tris pH 7.0 in a final volume of 700 µl. All the operations were performed at 4 °C.…”

Section: Reconstitution Of the Glutamine/amino Acid Transporter Into mentioning

confidence: 99%

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Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Oppedisano

Galluccio

Indiveri

2010

Biochemical Pharmacology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

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Section: Reconstitution Of the Glutamine/amino Acid Transporter Into mentioning

confidence: 99%

Section: Reconstitution Of the Glutamine/amino Acid Transporter Into mentioning

confidence: 99%

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Oppedisano

Galluccio

Indiveri

2010

Biochemical Pharmacology

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“…Protein eluates were reconstituted by removing the detergent with a hydrophobic column [13]. In this procedure, the mixed micelles containing detergent, protein and phospholipids were repeatedly passed through the same Amberlite XAD-2 column.…”

Section: Reconstitution Of the Ornithinelcitrulline Carrier In Liposomesmentioning

confidence: 99%

Identification and purification of the ornithine/citrulline carrier from rat liver mitochondira

Indiveri

Tonazzi

Palmieri

1992

European Journal of Biochemistry

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The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-1 00 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAESephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.In liver, two enzymes of the urea cycle, ornithine transcarbamylase and carbamyl phosphate synthetase I, are located in the mitochondrial matrix, while the other enzymes are cytoplasmic. Thus, for the operation of the urea cycle, ornithine must pass from the cytosol to the mitochondrial matrix where it is carbamylated to citrulline, and citrulline must leave the mitochondria in order to regenerate ornithine.The presence of an ornithine/citrulline carrier in the inner mitochondrial membrane has already been predicted in 1972 from theoretical and metabolic considerations [l]. However, the experimental evidence supporting the existence of this carrier in intact mitochondria is only circumstantial and not as definitive as for other mitochondrial metabolite carriers (for a review see [2 and 31). Based on the osmotic swelling of rat liver mitochondria, Gamble and Lehninger [4] [7], using rat liver mitochondria loaded with citrulline, presented evidence for the existence of an ornithine/citrulline-exchange system. These authors observed the uptake of ornithine in citrulline-loaded mitochondria and citrulline efflux from these mitochondria upon addition of ornithine or lysine. In a subsequent study 181, no evidence could be obtained for an ornithine/citrulline or lysine/citrulline exchange, whereas it was concluded that ornithine and lysine are taken up by rat liver mitochondria in exchange for H'. Recently, in rat kidney mitochondria, an The final proof for the existence of a particular carrier protein is its isolation. So far, an ornithine-transport protein has only been reconstituted into liposomes from a Lubrol WX extract of rat liver mitochondria [lo]. In this paper, we describe the isolation and purification of the ornithine/citrulline carrier from rat liver mitochondria using functional reconstitution as a monitor of the carrier activity during the isolation procedure. MATERIALS AND METHODS MaterialsHydroxyapatite (Bio-Gel HTP) was purchased from BioRad, Celite 535 from Roth, Amberlite XAD-2 from Fluka, DEAE-Sephacel, Sephadex PD-10, Sephadex G-50 and Sephadex G-75 from Pharmacia, L-[ ...

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“…These include DEAE cellulose or sepharose, gel filtration chromatography, hydroxylapatite, sucrose density gradients, etc. Furthermore, separation techniques have been developed for intrinsic membrane proteins, such as hydrophobic phase separation [121] and hydrophobic chromatography [122].…”

Section: Detergent-mediated Reconstitutionmentioning

confidence: 99%

Bacterial solute transport proteins in their lipid environment

Veld¹

1993

FEMS Microbiology Reviews

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Abstract:The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid species. Their activities are also sensitive to the lipid bilayer dynamics and physico-chemical state. Bacteria can adapt to changes in the environments (respective temperature, hydrostatic pressure, and pH) by altering the lipid composition of the membrane. Homeoviscous adaptation results in the maintenance of the liquid-crystalline phase through alterations in the degree of acyl chain saturation and branching, acyl chain length and the sterol content of the membrane. Homeophasic adaptation prevents the formation of non-bilayer phases, which would disrupt membrane organization and increase permeability. A balance is maintained between the lamellar phase, preferring lipids, and those that adopt a non-bilayer organization. As a result, the membrane proteins are optimally active under physiological conditions. The molecular basis of lipid-protein interactions is still obscure. Annular lipids stabilize integral membrane proteins. Stabilization occurs through electrostatic and possibly other interactions between the lipid headgroups and the charged amino acid residues close to the phospholipid-water interface, and hydrophobic interactions between the fatty acyl chains and the membrane-spanning segments. Reconstitution techniques allow manipulation of the lipid composition of the membrane in a way that is difficult to achieve in vivo. The physical characteristics of membrane lipids that affect protein-mediated transport functions have been studied in liposomal systems that separate an inner and outer compartment. The activity of most transport proteins is modulated by the bulk physical characteristics of the lipid bilayer, while specific lipid requirements appear rare.

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Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column

Cited by 111 publications

References 14 publications

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

Identification and purification of the ornithine/citrulline carrier from rat liver mitochondira

Bacterial solute transport proteins in their lipid environment

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