Membrane reconstitution

The rate of transport across bilayer membranes reconstituted from dipalmitoylphosphatidylcholine (DPPC) andnigericin is approximately the same as that observed across membranes reconstituted from DPPC and cecropin a at 35 C. Would you expect the transport rates across these two membranes also to be similar at 50 C Explain. 

Detergent removal during membrane reconstitution, Biochim. Biophys. Acta. 601, 328-342. 

Using the reconstitution approaches described above, we have demonstrated that phosphorylation of the skeletal muscle Ca channels by PKC results in activation of the channels [108], In the fluo 3-containing liposomes, channels phosphorylated by PKC exhibited a two-fold increase in the rate and extent of Ca " influx [108], Using the lipid bilayer-T-tubule membrane reconstitution system we are currently analyzing the effects of PKC-catalyzed phosphorylation at the single channel level [133], The demonstration that these channels undergo phosphorylation as a result of activation of PKC in intact skeletal muscle cells has not yet been achieved. 

Mayer D, Ataka K, Heberle J, Offenhaeusser A. 2005. Scanning probe microscopic studies of the oriented attachment and membrane reconstitution of cytochrome c oxidase to a gold electrode. Langmuir 21 8580-8583. 

Subsequent to possible solubilization of membrane-bound proteins, solubilization must be verified. The criteria listed in Table 2 are relevant in assessing whether solubilization has been accomplished. To ascertain whether the solubilized protein has retained biological activity, membrane reconstitution (28) is attempted subsequent to detergent removal (24). Reconstitution is often visualized by electron microscopy employing either negative staining or freeze fracture. 

Poste G, Nicolson G. Membrane Reconstitution, North Holland Publ. Co, Amsterdam, The Netherlands, 1982. 

Becker L et al. (2005) Preprotein translocase of the outer mitochondrial membrane reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol 353 1011-1020 Blobel G, Dobberstein (1975a) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67 835-851 Blobel G, Dobberstein (1975b) Transfer to proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol 67 852-862... 

Essentially, this method is based on subjecting cell membranes to a short external electric field pulse of an intensity comparable to the electric field strength of the membrane 77). Under these conditions the membrane breaks down locally and becomes permeable. This process is reversible, i.e. the membrane reconstitutes its original properties in time intervals which can be experimentally controlled. 

Transport by facilitated diffusion A large number of molecules and ions were shown to permeate membranes considerably faster than expected from their lipid-water partitioning behavior. This led to the recognition of additional transport mechanisms. Systematic investigations of permeability rates in membranes, reconstituted membranes, and membrane models as functions of the temperature of the nature and concentration of the permeant in the absence and in the presence of additives, suggested three different facilitated passive transport mechanisms ... 

We now know a great deal about the structure of cell membranes, ion pumps, electroporation, and membrane channels. In membrane reconstitution experiments, the evidence is that intracellular signal transduction begins at membrane receptors. The work... 

Lamb, J. D., West, J. N., Shaha, D. P., and Johnson, J. C. 2010. An evaluation of polymer inclusion membrane performance in facilitated transport with sequential membrane reconstitution. Journal of Membrane Science 365 256-259. 

FIGURE 1. Densitograms of spinach PSII proteins (a), PSII membranes (b), NaCl/urea-washed PSII membranes (c), NaCl/urea-washed membranes reconstituted with the 33-kDa protein at a protein to Chi ratio of 0.6 (w/w) (d), mixture of NaCl/urea-washed PSII membranes and the CH fragment added as a marker (e), NaCl/urea-washed PSII membranes (f), NaCl/urea-washed PSII membranes reconstituted with the CH fragment at a protein to Chi ratio of 0.6 (w/w). 

Constit. of Rhodiola sachalinensis. Non-ionic detergent solubilising agent for membrane proteins. Used in membrane reconstitution, and characterisation of enzymes, receptors, proteins - including human tumor necrosis factor (INF). Readily removed by dialysis. 

The purified CFP-Betlp contains degradation fragments. Most of these impurities are without transmembrane domains and do not incorporate into the liposomes they can be reproducibly removed during reconstitution. The CFP-Betlp protein after membrane reconstitution shows a single band in SDS-PAGE (Fig. 2). 

Fig. 2. Effect of membrane reconstitution on the Arrhenius plot of acetylcholinesterase from rats fed fat-free diet. Enz3mie in the original membrane ( ) in the solubilized state (o) and in the reconstituted vesicles (A). Initial velocity was expressed in an arbitrary scale.

A membrane reconstitution system was applied to investigate how the presence of CYPllAl affected CYP11B2 activity. Under steady-state reaction conditions, the interaction of CYP11B2 with CYPllAl stimulated the production of corticosterone from deoxycorticosterone but inhibited further reactions from corticosterone to aldosterone. This effect correlates also with the enhanced dissociation of corticosterone from CYP11B2 [34]. 

Membrane proteins can be oriented macroscopically, e.g. by reconstitution into lipid bilayers on glass plates which are aligned perpendicular to the magnetic field. Since all membrane-reconstituted proteins adopt the same orientation in lipid bilayers, narrow lines are observed, and their chemical shifts as well as their dipolar couplings are a signature of the orientation [1]. 

We have recently used polarized infrared (IR) spectroscopy to examine the extent and organization of protein structures in membrane associated proteins (4-6). In particular, with membrane reconstituted RC, we "ave determined the net orientation of thea -helical segments with respect to the membrane plane (6). In this article, we have made a comparison of the protein structures in the LMH and LM complexes in order to analyze the composition and the orientation of the secondary structures localized within the LM complex. We have investigated the UV circular di-chroism (CD) and polarized IR spectra of the LMH and LM complexes reconstituted in lipid vesicles. Our results show that transmembrane a-helices are present in both LMH and LM complexes. In addition, we also discussed some general features of membrane IR dichroism spectra. 

Darzon A, Vandenberg CA, Schonfeld M, Ellisman MH, Spitzer NC and Montal M (1980) Reassembly of protein-lipid complexes into large bilayer vesicles perspectives for membrane reconstitution, Proc.Natl.Acad.Sci USA 77, 239-243. Dutton PL, Mueller P, O Keefe DP, Packham NK, Prince RC and Tiede DM (1982) Electrogenic reactions of the photochemical reaction center and the ubiquinone-cytochrome b/c oxidoreductase, Curr.Top.Membr.Transp. 16, 323-343. Feher G and Okamura MY (1978) Chemical composition and properties of reaction centers. In Clayton RK and Sistrom WR, ed. The photosynthetic bacteria, pp. 349-386. New York Plenum Press. 

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