Proteoliposomes

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

A quantitative reconstitution approach was used to gain information as to the subunit composition of the -ATPase molecule [25]. Proteoliposomes prepared from asolectin and purified, radiolabeled ATPase molecules obtained by a freeze-thaw procedure similar to that of Dufour et al. [34] were shown to catalyze ATP hydrolysis-driven proton translocation, as indicated by the extensive quenching of aminochloromethoxyacridine fluorescence that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by... 

Unphosphorylated functioning according to Fig. 5 catalyzes facilitated diffusion of mannitol across the membrane. The same process has been reported for purified II reconstituted in proteoliposomes [70]. The relevance of this activity in terms of transport of mannitol into the bacterial cell is probably low, but it may have important implications for the mechanism by which E-IIs catalyze vectorial phosphorylation. It would indicate that the transmembrane C domain of Il is a mannitol translocating unit which is somehow coupled to the kinase activity of the cytoplasmic domains. We propose that the inwardly oriented binding site which is in contact with the internal water phase (Ecyt Mtl, see Fig. 5) is the site from where mannitol is phosphorylated when transport is coupled to phosphorylation. Meehan-... 

Kinetic measurements on II reconstituted in proteoliposomes are also consistent with the phosphorylation without transport. Il reconstituted by the detergent dialysis method into proteoliposomes assumes a random orientation the cytoplasmic domains face inward for 50% and outward for 50%. Those facing inward catalyze transport of external mannitol to the interior when E-I, HPr and P-enolpyr-uvate are included on the inside. Those facing outward convert external mannitol to external Mtl-P when HPr, E-I and P-enolpyruvate are included in the external medium. Comparison of the rates showed that the rate of external phosphorylation in this system was higher than the rate of transport. If transport and phosphorylation were obligatorily coupled, the rate of phosphorylation would not exceed the rate of transport [70]. 

Efflux of mannitol has been demonstrated using proteoliposomes containing only E. coli II . No II -dependent efflux of glucose from these proteoliposomes was observed. Furthermore, no efflux of mannitol was observed from liposomes lacking II " [70]. 

Babcock GJ, Mirzabekov T, Wojtowicz W, Sodroski J. Ligand binding characteristics of CXCR4 incorporated into paramagnetic proteoliposomes. J Biol Chem 2001 276(42) 38433-38440. 

It is emphasized that revealing the dynamics as well as the structure (or conformation) based on several types of spin-relaxation times is undoubtedly a unique and indispensable means, only available from NMR techniques at ambient temperature of physiological significance. Usually, the structure data themselves are available also from X-ray diffraction studies in a more refined manner. Indeed, better structural data can be obtained at lower temperature by preventing the unnecessary molecular fluctuations, which are major subjects in this chapter, since structural data can be seriously deteriorated for domains where dynamics are predominant even in the 2D or 3D crystalline state or proteoliposome at ambient temperature. It should be also taken into account that the solubilization of membrane proteins in detergents is an alternative means to study structure in solution NMR. However, it is not always able faithfully to mimick the biomembrane environment, because the interface structure is not always the same between the bilayer and detergent system. This typically occurs in the case of PLC-81(1-140) described in Section 4.2.4 and other types of peptide systems. 

Bloss, T., Clemens, S. and Nies, D. H. (2002). Characterization of the ZATlp zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes, Planta, 214, 783-791. 

Le Gaherec F, Bron P, Verbavatz JM, Garret A, Morel G, et al. 1996. Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection functional characterization of a novel aquaporin. J Gell Sci 109 (Pt 6) 1285. 

G. R. Borthiry, W. E. Antholine, J. M. Myers, and C. R. Myers, Addition of DNA to CrVI and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr111 complexes, Chem. Biodivers., 5 (2008) 1545-1557. 

Interaction of Drugs with Liposomes and Proteoliposomes Caroline Engvall and Per Lundahl... 

D. Chromatography of Drugs with Liposomes or Proteoliposomes as Interacting Phase... 

Several ILC studies covering drug interaction with liposomes and, correspondingly, proteoliposomes, cytoskeleton-depleted red blood cell membrane vesicles, red blood cell membranes, or red cells and ghosts have been reported (7,8,21-28,40,76,91,92,94). The log Ks values for interaction of... 

E Brekkan, Q Yang, G Viel, P Lundahl. Immobilization of liposomes and proteoliposomes in gel beads. In GF Bickerstaff, ed. Methods in Biotechnology. Vol. 1 Immobilization of Enzymes and Cells, Totowa, NJ Humana Press Inc, 1997, pp. 193-206. 

Haneskog, L., Zeng, C.-M., Lundqvist, A., and Lundahl, P, Biomembrane affinity chromatographic analysis of inhibitor binding to the human red cell nucleoside transporter in immobilized cells, vesicles and proteoliposomes. Biochim. Biophys. Acta, 1371, 1-4, 1998. 

Figure 27. Comparison of the L-Clu-induced integrated channel currents between two different preparations of GluR-incorporated BLMs upon injecting the identical concentration of l-GIu (0.10 nM). Applied potential +50 mV. Conditions 9.8 mM HEPES-NaOH (pH 7.6) containing 0.52 M NaCI, 0.19 mM CaCl2, 4.8 mM glycine, 24 pg mL of concanavalin A, 8.1 mM sucrose and 0.40 M formamide in both cis and trans side solutions. Proteoliposomes were injected only to the cis side and agonist solution was added to the trans side. Applied potential +50 mV. The l-GIu solution was injected to the trans side. ...

The incorporation of purified Na /D-glucose cotransporter into the BLM formed by the monolayer folding method was achieved either by fusion of its proteo-liposomes or by folding the lipid layer containing the proteoliposomes. The experimental setup for measuring the currents is the same as in Figure 25. The concentration dependence of the observed currents is shown in Figure 31. A Na ... 

For -LCAT activity the apoA-I proteoliposome emulsion is prepared by evaporating 260 pi of 5 mg/ml egg yolk phosphatidylcholine, 150 pi of 1 mg/ml unesteri-fied cholesterol, and 3 pi of 21 Ci/mmol [7-3H(N)]-cholesterol. The dried lipids are dissolved in 125 pi pure ethanol and injected into 10 ml of analysis buffer and vor-texed. The emulsion is concentrated by ultrafiltration to less than 2.5 ml and then filled up to 2.5 ml. A 300-pL aliquot of this emulsion is incubated with 75-150 mg of apoA-I and 1.1 ml analysis buffer. The optimal amount of apoA-I varies from lot to lot and has to be optimized using normal plasma samples. 

For the analysis, 140 pi of the apoA-I proteoliposomes is supplemented with 50pl of 8% BSA in analysis buffer, 10 pi of 0.1 M 2-mercaptoethanol, and 15 pi plasma. The mixture is incubated by shaking for 30 min at 37°C. The LCAT reaction is stopped by adding 4 ml of chloroform methanol (2 1, v/v). Lipids are extracted by incubation at room temperature for 2 h. Hydrophilic and lipophilic phases are separated by adding... 

To prepare proteoliposomes, 7 mg egg yolk phosphatidylcholine, 1.16 pg cholesterol, 77.5 pg cholesteryl oleate and 10 pCi [3H]-cholesteryl oleate, all dissolved in chloroform are mixed. After evaporation of chloroform with nitrogen, the lipids are resolved in 400 pi ethanol. The ethanolic solution is injected into 5 ml of a vortex-ing buffer with 39mmol/l sodium phosphate, 0.01% EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) 3 mg apoA-I is added. The solution is subsequently dialyzed against a buffer with 39 mmol/1 sodium phosphate, 0.01 % EDTA, 2 mmol/1 NaN3, and 12 mmol/1 sodium cholate (pH 7.4) at 4°C. At the end the solution is filled up with analysis buffer containing 39 mmol/1 sodium phosphate,... 

Counts per minute (CPM)totli = CPM in blank sample (or CPhdtotal x 10 in ml proteoliposomes)... 

The incorporation of a membrane protein into a polymerizable liposome from (22) was demonstrated by R. Pabst n9). The chromoprotein bacteriorhodopsin — a light-driven proton pump from halophilic bacteria — was incorporated into monomeric sulfolipid liposomes by ultrasonication. The resulting proteoliposomes were poly-... 

The Na+ gradient established by the (Na+, K+)-ATPase is utilized in the transport of a number of solutes into animal cells via Na-solute cotransport or symport.68 This is well known for glutamate. Renal Na+ cotransport systems for mono- and di-carboxylic acids and for various amino acids have been functionally reconstituted in proteoliposomes. These transport systems are polypeptides solubilized from brush border membranes of renal proximal tubules. The establishment of Na+ gradients (high Na+ outside) resulted in increases in concentrations of substrate inside the proteoliposomes.69... 

When reconstituted into proteoliposomes with Ml muscarinic cholinergic receptor, PLC/11 potently stimulates the GTPase activity of Gaq, achieving a steady-state rate 1000-fold greater than that in the absence of a GAP... 

I.J. Ryrie and N. Fuad, Membrane adhesion in reconstituted proteoliposomes containing the light-harvesting chlorophyll a/b-protein complex the role of charged surface groups, Arch. Biochem. Biophys. 214 (1982) 475-488. 

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