Translocators liposomes

Ghosh, S., Xie, W.Q., Quest, A.E.G., Strum, J.C. and Bell, R.M., 1994, The cystein-iich region ofraf-lkinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras./.Biol. Chem.269 10000-10007. 

Figure 1 Potential points for the enhancement of liposome-mediated gene transfer. The above diagram illustrates the characteristic lipofection pathway demonstrating the four key stages bold, underlined), complex formation, targeting and internalization, endosomal escape, and nuclear translocation. Indicated alongside (italic) are the peptides that can be used to augment the transfection potential of the liposome. Abbreviation pDNA, plasmid DNA.

TAT liposomes remain intact within one hour of translocation and slowly migrate through the cell, bypassing the endocytic pathway, to the perinuclear zone where they disintegrate (95). The mechanism utilized by TAT to migrate across the membrane was thought to be energy independent because it operates at similar rates at both 4°C and 37°C (95,96). Cell entry by TAT is also unhindered by metabolic inhibitors such as sodium azide or iodoacetamide (97). Peptides constructed of both the d and l amino acids of Antp can be detected intracellularly, the inference of which is that no specific receptor was required because both isomers had equal potential (98,99). 

The amino acid sequences of haptides comprise hydrophobic and cationic residues with a net charge of +4 to +5 per 19 to 21 amino acids. It was proposed that haptides could be attracted to the anionic liposomes as well as the anionic cell membrane and that the hydrophobic properties of the haptide facilitate membrane translocation (106). Haptide uptake was reported to be energy independent, occurring at 4°C. The advantage of this peptide compared to CPP such as TAT and Antp, is that, unlike the virus-derived peptides, the haptides are not recognized as foreign antigens and do not induce cell transformation (106). However, haptides have also been found to accelerate fibrin clot formation and lack cell specificity (106). 

The Mg2+-activated ATPase (or ATP synthase) is made up of two parts. The Fj component is the catalytic, Mg2+-binding, extrinsic membrane protein composed of five different subunits, a, (3, y, S and e. The F0 component is an intrinsic membrane complex that contains three subunits, a, b and c, and mediates proton translocation. The F, protein is bound to the membrane through interaction with F0. The complexity of the F,F0 enzyme has presented many difficulties. Hie greatest advances have been made for the bacterial enzymes, notably for thermophiles, Escherichia coli and Rhodospirillum rubrum, where progress has been made in the purification of subunits and their reconstitution into membranes, and the identification of binding sites for Mg2+ and nucleotides on the Fi subunits.300 FiF0 preparations can be incorporated into liposomes and display H+ translocation, ATP-P, exchange and ATP synthesis.301... 

Perhaps more important than all these considerations was the fact that preparation of functional vesicular systems led to purification studies and reconstitution of transporters using artificial liposomes and imposed ion gradients to drive translocation. Vesicle systems also bypassed the need to measure binding as the one means... 

The transition from coated vesicle to early endosome is accompanied by acidification of the vesicular lumen that continues into the late endosomal and lysosomal compartments, reaching a final pH in the perinuclear lysosome of approximately 4.5. Such acidification associated with endosome maturation provides the means by which certain viruses gain access to the cytosol. Acid-induced conformational changes in the viral proteins trigger translocation across the endosomal membrane via a fusion process. By taking advantage of the endosomal acidification, pH-sensitive liposomes, adenovirus and endosomolytic peptides have been used to facilitate the release of plasmids into the cytoplasm prior to lysosomal degradation. 

Tseng, Y. L., Liu, J. J., and Hong, R. L. (2002) Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat a kinetic and efficacy study. Mol. Pharmacol. 62, 864-872. 

The isolated CFq-CFi has been incorporated into phospholipid liposomes and shown to carry in this form most of the energy-transducing functions which it catalyses within the thylakoid membranes. Thus, the reconstituted ATP synthase carries out ATP-dependent proton translocation resulting in both a 4pH and a developing across the reconstituted liposomes [72,73] an uncoupler-sensitive ATP-Pj exchange reaction [39] and ATP formation driven by artificially imposed 4pH and Ail/ [39,74,75], or by electric field pulses [56]. The ATP synthase proteolipo-somes provide the simplest system available today for the study of electrochemical-gradient-driven phosphorylation. 

In this account we will review the structural and functional properties of the respiratory chain components, as they are known from studies with intact mitochondria, vesicles of the inner mitochondrial membrane (submitochondrial particles), or isolated complexes. The latter may additionally be reconstituted into liposomal membranes. To some extent we will also review the knowledge on the integrated functions of the respiratory chain with main emphasis on proton translocation and essential thermodynamic and kinetic properties. 

Comparatively few papers describe the properties of Complex III after reconstitution into liposomal membranes [198,279,280]. The found stoicheiometries of proton and electrical charge translocation agree well with the data for mitochondria. 

Ragan and Hinkle [42) reported on proton translocation by Complex I reconstituted into liposomes. During oxidation of added NADH by ubiquinone-1 they observed an uptake of about 0.7 H /e in addition to the trivial proton uptake linked to reduction of quinone by NADH. Liposomes reconstituted with Complex I plus ATP synthase exhibited ATP synthesis linked to oxidation of NADH by Q-1 [311]. 

Two major ATP synthesizing reactions in living organisms are oxidative phosphorylation and photophosphorylation. Both reactions take place in H -ATPase (FqF,), which is driven by an electrochemical potential difference of protons across the biomembrane, as predicted by Mitchell [1]. In Racket s laboratory, ATPases related to oxidative phosphorylation were prepared, but their relationship to Mitchell s chemiosmotic hypothesis [1] was not described [2], Later, an insoluble ATPase (H -ATPase) was shown to translocate protons across the membrane when it was reconstituted into liposomes [3], H -ATPase was shown to be composed of a catalytic moiety called F, (coupling factor 1) [4], and a membrane moiety called Fq [5], which confers inhibitor sensitivity to F,. F was shown to be a proton channel, which translocates down an electrochemical potential gradient across the membrane when Fg is reconstituted into liposomes (Fig. 5.1) [6]. Thus, -ATPase was called FqFj or ATP synthetase. 

Proton motive ATP synthesis in FqF, liposomes (Fig. 5.1, Table 5.4) and AjEtH -driven translocation through Fg [6] strongly supported Mitchell s chemiosmotic theory described in the previous series of Comprehensive Biochemistry [108]. The molecular mechanism of function of FgF, was not predicted by this theory, but it has been elucidated by new methods. For example, the presence of X-P was demonstrated in... 

The function of bacteriorhodopsin as a light-driven proton pump is well established from studies [14,70,83-85,323] of whole H. halobium cells, cell envelope vesicles prepared from the cells [78,324], and liposomes [17,18,135,191,325-327] as well as planar films [328-339] into which purple membrane was incorporated. In all of these cases light-dependent net translocation of protons across the membrane is observed, whose magnitude exceeds the number of bacteriorhodopsin molecules in the system by up to two orders of magnitude. 

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