ATP-liposomes

ATP cannot be effectively delivered to most tissues including the ischemic myocardium without protection from degradation by plasma endonucleotidases. However, it has been established that ATP can be delivered to various tissues by its encapsulation within liposomal preparations. We describe here, the materials needed and methods used to optimize the encapsulation of ATP in liposomes, enhance their effectiveness by increasing their circulation time and target injured myocardial cells with Uposomal siufece anti-myosin antibody. Additionally, we outline methods for ex vivo studies of these ATP liposomal preparations in an isolated ischemic rat heart model and for in vivo studies of rabbits with an induced myocardial infarction. The expectation is that these methods will provide a basis for continued studies of effective ways to deliver energy substrates to the ischemic myocardium. 

Key words ATP, Liposomes, Immunoliposomes, Antimyosin, Ischemia, Isolated rat heart. Rabbit myocardial infarction... 

Key words Ischemia, Hypoxia, Intracellular delivery, ATP, Liposome, Energy, Endothelial cells,... 

Figure C3.2.17. Diagram of a liposome-based artificial photosynthetic membrane showing the photocycle that pumps protons into the interior of the liposome and the CFqF j-ATP synthase enzyme. From [55],...

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-... 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

One problem with the ATP assay in aqueous media is that the enzyme requires hydrophobic media the reaction rate of luciferase-catalyzed reactions is variously affected by the presence of detergents [117, 118]. The presence of cationic liposomes improves sensitivity by a factor of five times compared to that in water alone [119]. 

However, subsequent studies demonstrated that the formation of hydroxyl radicals, even if it takes place during lipid peroxidation, is of no real importance. Beloqui and Cederbaum [11] have found that although the glutathione-glutathione peroxidase system suppressed hydroxyl radical generation during the oxidation of 4-methylmercapto-2-oxo-butyrate, it exhibited a much smaller effect on microsomal lipid peroxidation. Therefore, hydroxyl radical formation is apparently unimportant in this process. Other authors also pointed out at an unimportant role of hydroxyl radicals in the initiation of microsomal lipid peroxidation [12 14], For example, it has been shown that Fe(EDTA), a most efficient catalyst of hydroxyl radical formation by the Fenton reaction, inhibited microsomal and liposomal lipid peroxidation, while the weak catalysts of this reaction Fe(ADP) and Fe(ATP) enhanced it [13]. 

In the early 1960s Bangham prepared liposomes—sealed vesicles made from lecithin or other lipids, with an aqueous interior. Liposomes with purified Na/K-ATPase inserted into their membranes pumped cations in the presence of ATP, convincingly confirming the function of the ATPase, and the existence of vectorially (asymmetrically) organized proteins. 

Figure 11.9 Schematic view of the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes, (i) Freeze-thaw (x 7) solution containing t-RNAP , poly(U), Phe, ATP, GTP, Mg(OAc)2, NH4CI, spermine, spermidine, phos-phoenolpyruvate. (ii) Soution containing pyruvate kinase, 100 000 g supernatant enzymes, 308 and SOS ribosomal subunits, (iii) 1. Free-thaw (x3) 2. Brief extrusion 3. Addition of EDTA (final concentration = 35 mM. (iv) Withdrawl of aliquots at indicated time and cold TCA precipitation. Analysis of the radioactivity remaining on the glass filter by p-scintillation counting. (Modified from Oberholzer et al, 1999.)...

The common strategy is to entrap into the aqueous core of liposomes all the ingredients for the in vitro protein expression i.e., the gene for the GFP (a plasmid), an RNA polymerase, ribosomes, and all the low-molecular-weight components (amino acids, ATP, etc.) needed for protein expression. 

A crucially important finding is that submitochon-drial particles or vesicles from broken chloroplasts will synthesize ATP from ADP and P , when an artificial pH gradient is imposed.172186 Isolated purified FjF0 ATPase from a thermophilic Bacillus has been coreconstituted into liposomes with the light-driven proton pump bacteiiorhodopsin (Chapter 23). Illumination induced ATP synthesis.187 These observations support Mitchell s proposal that the ATP synthase is both spatially separate from the electron carriers in the membrane and utilizes the protonmotive force to make ATP. Thus, the passage of protons from the outside of the mitochondria back in through the ATP synthase induces the formation of ATP. What is the stoichiometry of this process ... 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

Fig. 41. ATPase activity of ATP synthetase incorporated into liposomes (-O-) incubation of the enzyme with polymerized liposomes (-+-) incubation of the enzyme with monomeric liposomes of (22) followed by polymerization (- ) activity of ATP synthetase in soybean lecithin liposomes24. ATPase activity was measured in 100 mM Tris-HCl (pH 8.0) in the presence of 1 mM ATP at 37 °C by determining the liberated orthophosphate...

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... 

Many of the metabolite uptake studies cited above rely on combined uptake and incorporation into starch. In order to separate uptake from incorporation, Schott et al.226 extracted amyloplast membrane proteins from potato tubers and reconstituted them into liposomes. These reconstituted liposomes transported Pi, triose phosphates and G6P in a counter-exchange mode. The liposomes were ineffective in the transfer of G1P uptake of ADP-Glc was not tested. Mohlmann et al.236 have used a proteoliposomic system to reconstitute plastid envelope proteins. In this system, ADP-Glc is transported in exchange for AMP. Thus the more widely studied plastid ATP/ADP transporter was not responsible for ADP-Glc uptake. More recently, Bowsher et al.237 reported that wheat endosperm amyloplasts membrane proteins reconstituted into proteoliposomes took up ADP-Glc in exchange for AMP and ADP. In addition, they showed that under conditions of ADP-Glc dependent starch biosynthesis, the efflux of ADP from intact amyloplasts was equal to that of ADP-Glc utilization by starch synthesis. The amyloplast membrane ADP-Glc/ADP transporter was a 38 000 molecular weight integral membrane protein.237... 

Reconstituted liposomes (6 pg protein) were suspended in buffer containing 1 pM acridine orange (A) or 1 pM oxonol V (B), and fluorescence was monitored. At indicated times, 1 mM ATP, 0.1 pM valinomycin and 1 pM CCCP were added.32 ... 

Although remarkable progress has been made in matching the secondary structure and the function in the various members of the mitochondrial carrier family (del Arco and Satrustegui 2005), satisfying information about the function of potential ADP/ATP transporters can only come from a detailed functional analysis (see later). Several methods are currently available reconstitution in liposomes, in vivo expression, rescue experiments, and functional tests in bacteria (Escherichia coli, Lactococcus lactis). Such tests involve competition experiments, effector and inhibitor studies (Voncken 2001 Voncken et al. 2002a Haferkamp et al. 2002 van der Giezen et al. 2002 Tjaden et al. 2004 Chan et al. 2005 Leroch 2006). 

Frith JC, Monkkonen ), Blackburn GM, Russell RGG, Rogers M). Clodronate and liposome-encapsulated clodronate are metabolised to a toxic ATP analog, adenosine 5 -(/ / ,yy-dichloromethylene) triphosphate, by mammalian cells in vitro. J. Bone Miner. Res., 1997, 12, 1358-1367. 

Figure 11.4. Schematics of calcium transport with Ca2+-ATPase liposome, ionophore (A), and leaks, lonophore A23187 induces uptake of Ca2+ions. Leaks are shown with dashed arrows, i is interior (alkaline) and e is exterior (acidic). Scalar flow of ATP hydrolysis...

Tlirina, P, Samoray, D., and Graber, P. 2003. H7ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalyzed by CFoF1-liposomes. EMBO J. 22 418-426. 

The ATP-P exchange reaction has been very useful in demonstrating the activity of the complete ATP synthase (CFy-CFi) complex, since it requires an intact system properly incorporated within a vesicular system [39]. Thus, isolated CFi or CFo-CFi, though possessing ATPase activity, do not show ATP-Pj exchange until properly incorporated into a liposomal system. 

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. 

Several triads (as well as more complex systems, tetrads, pentads, etc.) have been successfully developed making use of organic molecular components [211-213], some of which (porphyrins, quinones, carotenoids) are reminiscent of those found in the natural systems (for detailed accounts, see Volume III, Part 2, Chapters 1 and 2). Remarkable efficiencies and lifetimes of charge separation have been reached with such systems. Their potential has been impressively demonstrated by incorporation into liposomal membranes performing the photo-driven synthesis of ATP [214]. 

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