High‐performance liquid chromatography of the main polypeptide (MP26) of lens fiber plasma membranes solubilized with <i>n</i>‐octyl β‐D‐glucopyranoside

Manenti, Stéphane; Dunia, I.; Maire, Marc le; Benedetti, E.L.

doi:10.1016/0014-5793(88)81373-5

Cited by 15 publications

(9 citation statements)

References 16 publications

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“…The molecular weight of 89,000 includes an appreciable contribution from bound detergent. Calculations based on the assumption of a protein-detergent complex with detergent bound at levels of 1 g/g protein (13,35) imply a contribution of approximately M r 60,000 for the protein component alone, indicating that BtuB was purified as a monomer. Similar calculations with estimates of detergent bound at a ratio of 0.5 g/g (approximately the molecular weight of a detergent micelle) or 1.5 g/g protein result in only a 20% difference in the calculated molecular weight, which is consistent with the conclusion that purified BtuB is monomeric.…”

Section: Resultsmentioning

confidence: 99%

“…The partial specific volume of BtuB (0.726 cm 3 /g) was calculated from the amino acid composition. A partial specific volume of 0.859 cm 3 /g was used for octylglucoside (35). Molecular masses were generated for both the protein-detergent complex and the protein component alone, estimating the detergent bound to the protein at 0.5, 1.0, and 1.5 g/g (35).…”

Section: Methodsmentioning

confidence: 99%

“…A partial specific volume of 0.859 cm 3 /g was used for octylglucoside (35). Molecular masses were generated for both the protein-detergent complex and the protein component alone, estimating the detergent bound to the protein at 0.5, 1.0, and 1.5 g/g (35). Sedimentation velocity experiments were conducted as described above after the preincubation of BtuB (0.5 mg/ml) with equimolar levels of colicin E3 to directly demonstrate the formation of the BtuB-colicin E3 complex.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Purification and Characterization of Monomeric Escherichia coli Vitamin B12 Receptor with High Affinity for Colicin E3

Taylor¹,

Burgner²,

Clifton³

et al. 1998

Journal of Biological Chemistry

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The btuB gene product from Escherichia coli is a 66.5-kDa integral outer membrane protein required for highaffinity uptake of cyanocobalamin and the translocation of E group colicins and colicin A. Efficient purification of overexpressed BtuB containing stoichiometric levels of bound lipopolysaccharide has been achieved through the extraction of the outer membrane with nonionic detergent followed by ion-exchange chromatography. Analysis of far UV circular dichroism spectra indicates a predominantly ␤-sheet secondary structure (76 ؎ 4%) with a low ␣-helical content (15 ؎ 3%), providing the first direct evidence for secondary structure models derived from sequence and hydropathy analysis. Characterization of the octylglucoside-solubilized receptor by sedimentation equilibrium and sedimentation velocity analysis reveals a monodisperse protein-detergent complex of approximately 89 kDa with a sedimentation coefficient of 4.7 S which, after correction for bound detergent, indicates that BtuB is purified as a monomer. BtuB binds vitamin B 12 with a stoichiometry of approximately 1:1, as observed by a shift in the sedimentation profile of the vitamin to the much faster velocity observed for the protein-detergent complex. The preincubation of colicin E3 with stoichiometric levels of BtuB protects susceptible strains from the lethal effects of the colicin and results in a complex with a sedimentation coefficient appropriate for a BtuB-detergent-colicin E3 complex, demonstrating that monomeric BtuB retains high affinity for this particular ligand after isolation.The outer membrane of Escherichia coli and other Gramnegative bacteria has evolved to exclude harmful agents present in the surrounding environment while maintaining the ability to internalize required nutrients of low abundance. Although general barrier function is largely provided by the lipopolysaccharide comprising the outer leaflet of this asymmetric bilayer (1, 2), the complexities of nutrient uptake are addressed by a variety of integral membrane proteins that operate by passive diffusion, facilitated diffusion, or active transport (3, 4). Bacterial porins form trimeric, aqueous-filled channels that direct passive or facilitated diffusion of hydrophilic compounds up to a molecular weight limit of approximately 600 (3). Extensive biochemical and electrophysiological characterization (5), together with high resolution structure determination of both porin types (6, 7), has provided insight into the manner in which a membrane-spanning ␤-barrel architecture serves to overcome the energetic problems associated with the transport of polar solutes through the membrane bilayer. The comparison of passive diffusion channels with those functioning by facilitated diffusion illustrates the nature of modifications to the basic porin fold that confer specificity to the transport process (6).The acquisition of vitamin B 12 and iron-bearing siderophores presents additional complications for E. coli cells due to their relative low abundance and molecular masses that exceed th...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Purification and Characterization of Monomeric Escherichia coli Vitamin B12 Receptor with High Affinity for Colicin E3

Taylor¹,

Burgner²,

Clifton³

et al. 1998

Journal of Biological Chemistry

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“…A monomer-tetramer equilibrium has been demonstrated previously. 23,24 In summary, it appears that different oligomeric forms of solubilized AQP0, up to octamers, are in an equilibrium that is largely dependent on the experimental conditions.…”

Section: Biophysical Characterizationmentioning

confidence: 98%

Co-axial Association of Recombinant Eye Lens Aquaporin-0 Observed in Loosely Packed 3D Crystals

Palanivelu

Kozono

Engel

et al. 2006

Journal of Molecular Biology

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“…In normal lens fibers and in reconstituted proteoliposomes containing exclusively MP26, the protein forms tetrameric oligomers randomly dispersed in the bilayer (19). Furthermore, the MP26 oligomers can form, both in situ and in reconstituted proteoliposomes (19,46), square arrays of tetrameric subunits. However, in normal lenses, the lattice organization is restricted to the plasma membranes of the nuclear fibers (3,13,22,39,43).…”

Section: Ultrastructural Features Of the Lens Expressing Mdr3-pgpmentioning

confidence: 99%

Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract.

Dunia

Smit

Valk

et al. 1996

The Journal of Cell Biology

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Abstract. We have generated mice transgenic for a human multidrug resistance (MDR)3 mini-gene driven by a hamster vimentin promoter. The MDR3 gene encodes a P-Glycoprotein that resembles the mouse multidrug resistance 2 P-Glycoprotein shown to be involved in the translocation of the phospholipid phosphatidylcholine through the hepatocyte canalicular membrane (Smit et al., 1993. Cell. 75:451-462). The vimentin promoter drives expression of the MDR3 transgene in mesenchymal tissues and in the eye lens.We show here that the presence of human multidrug resistance 3 P-Glycoprotein in the lens results in a severe lenticular pathology. Lens structural abnormalities initiate at a late embryonic stage and increase during postnatal lens development. Differentiation of the primary fibers is affected, and the terminal differentiation of the lens epithelium into secondary fibers is also perturbed. The ultrastructural alterations, particularly of the lens plasma membranes, resemble those identified in congenital mouse osmotic cataract. p -GLYCOPROTEINS (pgp)l are highly conserved membrane proteins that can function as ATP-dependent efflux pumps (23,31,57). They belong to the family of ATP-binding cassette transporter proteins (37). Two genes for Pgps have been identified in humans: human multidrug resistance gene (MDR)I and MDR3 (also called MDR2 [10,72,73], and three in mice: mouse multidrug resistance gene (mdr)l (or mdrlb), mdr3 (or mdrla), and mdr2 (16,33,35).The human multidrug resistance (MDR)I Pgp (and the related murine mdrl and mdr3, and hamster pgpl and pgp2 Pgps), can extrude a wide range of hydrophobic drugs from mammalian cells (18,34,42,70 pounds may represent the main physiological function of these Pgps (60). Attempts to show that the human MDR3 or the closely related mouse mdr2 Pgp (91% identity at the amino acid level) can confer MDR have been negative thus far (5,35,58,73).To find a physiological function for this class of Pgps, we have generated mice that are either unable to make the mdr2 Pgp or overproduce the MDR3 Pgp in many tissues (64; Smit, J.J.M., F. Baas, J.E. Hoogendijk, G. Jansen, F. Jennekens, M.A. van der Valk, A.H. Schinkel, A.J.M. Berns, K. Nooter, and P. Borst, manuscript in preparation). Mice homozygous for a disrupted mdr2 gene develop liver disease. A detailed analysis of these mice has shown that the mouse mdr2 Pgp (and presumably therefore also its human MDR3 counterpart) is essential for translocating phosphatidylcholine through the hepatocyte canalicular membrane into the bile (64,65). This indicates that this Pgp is a specific phospholipid translocator, as has recently been supported by in vitro experiments (54, 66).We have generated mice containing an MDR3 minigene under the control of a vimentin promoter which drives the expression of the transgene in mesenchymal tissues and in the eye lens. These mice develop a peripheral neuropathy and microphthalmia. Here we present an analysis of the abnormalities in the lenticular cells of these

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High‐performance liquid chromatography of the main polypeptide (MP26) of lens fiber plasma membranes solubilized with n‐octyl β‐D‐glucopyranoside

Cited by 15 publications

References 16 publications

Purification and Characterization of Monomeric Escherichia coli Vitamin B12 Receptor with High Affinity for Colicin E3

Purification and Characterization of Monomeric Escherichia coli Vitamin B12 Receptor with High Affinity for Colicin E3

Co-axial Association of Recombinant Eye Lens Aquaporin-0 Observed in Loosely Packed 3D Crystals

Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract.

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