scholarly journals Production of Plasma Membrane Vesicles with Chloride Salts and Their Utility as a Cell Membrane Mimetic for Biophysical Characterization of Membrane Protein Interactions

2012 ◽  
Vol 84 (20) ◽  
pp. 8650-8655 ◽  
Author(s):  
Nuala Del Piccolo ◽  
Jesse Placone ◽  
Lijuan He ◽  
Sandra Carolina Agudelo ◽  
Kalina Hristova
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Protein Interactions ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Biophysical Characterization ◽  
Membrane Mimetic ◽  
A Cell ◽  
Chloride Salts

scholarly journals Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi

1995 ◽  
Vol 306 (1) ◽  
pp. 299-303 ◽  
Author(s):  
G Benaim ◽  
S N J Moreno ◽  
G Hutchinson ◽  
V Cervino ◽  
T Hermoso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
High Sensitivity ◽  
Bovine Brain ◽  
Calcium Pump ◽  
Plasma Membrane Vesicles ◽  
P Type

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

Synthesis of NTA3-DTDA — A Chelator-Lipid that Promotes Stable Binding of His-Tagged Proteins to Membranes

2006 ◽  
Vol 59 (5) ◽  
pp. 302 ◽  
Author(s):  
Joseph G. Altin ◽  
Martin G. Banwell ◽  
Phillip A. Coghlan ◽  
Christopher J. Easton ◽  
Michael R. Nairn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Targeted Delivery ◽  
Membrane Vesicles ◽  
Spectroscopic Characterization ◽  
Plasma Membrane Vesicles ◽  
Reaction Sequence ◽  
Type Compound

A six-step reaction sequence is described for the preparation of compound 1 (NTA3-DTDA), a membrane-penetrating and potent chelator that can be incorporated into liposomes and plasma membrane vesicles containing antigens and thus allowing targeted delivery of such assemblies to a variety of cells for the purposes of eliciting anti-tumour responses. Full spectroscopic characterization of this dendritic-type compound as well as certain of its precursors is reported.

scholarly journals HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy

2020 ◽  
Vol 295 (45) ◽  
pp. 15183-15195 ◽  
Author(s):  
Amanda E. Ward ◽  
Volker Kiessling ◽  
Owen Pornillos ◽  
Judith M. White ◽  
Barbie K. Ganser-Pornillos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Membrane Fusion ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Lipid Binding ◽  
Plasma Membrane Vesicles ◽  
Tirf Microscopy ◽  
Host Restriction ◽  
Whole Cells

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

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