[17] Isolation of integral membrane proteins by phase partitioning with triton X-114

Brusca, John S.; Radolf, Justin D.

doi:10.1016/0076-6879(94)28019-3

Cited by 140 publications

(111 citation statements)

References 40 publications

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“…The supernatant was sedimented at 100 000 × g for 3 h at 4ЊC, giving rise to soluble (S100) and particulate (P100) fractions. P100 was resuspended by homogenization in 6 ml icecold TBS, while 1 ml S100 was extracted utilizing Triton X-114 (Sigma) (10% stock solution) at a final concentration of 1% or 2%, as described previously (Bordier, 1981;Brusca and Radolf, 1994). The detergent phase was diluted with TBS to a final volume of 0.5 ml.…”

Section: Purification Of Haemolysin From E Colimentioning

confidence: 99%

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Guina

Oliver

1997

Molecular Microbiology

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SummaryWe cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted ␣-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.

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Section: Purification Of Haemolysin From E Colimentioning

confidence: 99%

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Guina

Oliver

1997

Molecular Microbiology

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“…To examine the hydrophobicity of Bax, Triton X-114 phase partitioning was used (20,21). Briefly, cytosolic extracts prepared in isotonic buffers at either pH 6.9, 7.8, or 8.1 were treated with 2% (vol͞vol) Triton X-114 (Sigma), and the hydrophobic detergent phase was partitioned by incubation at 37°C followed by high-speed centrifugation.…”

Section: Cellsmentioning

confidence: 99%

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

Khaled

Kim

Hofmeister

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

228

241

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IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

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“…The derivation and characterization of the CD1b-restricted, mycobacterial lipid Ag-specific T cell lines DN1 and LDN5, and of the CD1b-restricted ganglioside-reactive T cell clones GG123B and GG33A, have been previously described in detail (11,12,28). The CD1b-specific autoreactive Mt2.21 T cell clone was derived from the CD4-depleted PBMC of a randomly selected normal blood donor by in vitro stimulation with CD1 ϩ monocyte-derived dendritic cells (DC 4 ; produced as described below) in T cell medium (TCM) (8) containing 10 g/ml of a detergent extract from Mycobacterium tuberculosis (29). These cells were subsequently cloned by limiting dilution and expanded by PHA stimulation.…”

Section: Cell Linesmentioning

confidence: 99%

Molecular Recognition of Human CD1b Antigen Complexes: Evidence for a Common Pattern of Interaction with αβ TCRs

Melián

Watts

Shamshiev

et al. 2000

The Journal of Immunology

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Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the α1 and α2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the α helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both α helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.

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[17] Isolation of integral membrane proteins by phase partitioning with triton X-114

Cited by 140 publications

References 40 publications

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

Molecular Recognition of Human CD1b Antigen Complexes: Evidence for a Common Pattern of Interaction with αβ TCRs

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