ATPase complex reconstitution

The oligomycin-sensitive ATPase complex, a major enzyme of the mitochondrial inner membrane, can serve to illustrate some of these principles. The ATPase complex is a water-insoluble enzyme which spontaneously associates into vesicular membranes in the presence of phospholipids. The ultrastructural appearance of the reconstituted ATPase membranes is similar to that of the native inner mitochondrial membrane. The complex consists of at least 10 different subunit polypeptides which have been resolved into 3 components, each with a measurable function (1) Five of the polypeptides are part of a catalytic unit called Fi, a water-soluble ATPase which neither requires phospholipids for activity nor has any detectable capacity for binding phospholipids. (2) Four other polypeptides form a unit which has no presently known enzymatic function, but when combined with Fi modifies its physical and catalytic properties. The proteins of this unit are extremely insoluble in water and can combine with phospholipids to form membranes. One of the proteins of the membrane unit is characterized by an unusually large proportion of nonpolar amino acids and a high affinity for phospholipids. (3) The third component (OSCP) is a single polypeptide whose function is to link F to the hydrophobic membrane unit. In the purified state, this protein is completely water soluble. ... 

The proton-translocating ATPase complex has been the object of many studies tc elucidate its energy-transducing function in the direction of both ATP synthesis and hydrolysis. By reconstitution in liposomes, mostly prepared from aso-lectin, a simple and functional model system can be obtained. Recently, a DCCD-sensitive H -ATPase complex has been isolated from the thermophilic cyanobacterium SyneohoooQOUS 6716 (Lubberding et al., submitted) as a Mg " -dependent heat-stable complex, which can only be activated by trypsin. This complex, reconstituted with liposomes from isolated Synechocooous lipids catalyzes DCCD-sensitive exchange and ATP hydrolysis activities and ge-... 

TABLE 1. Catalytic activities of the isolated and reconstituted Syneohooooous 6716 ATPase complex. The lipid/protein ratios of the reconstituted liposomes were 0.01 (w/w). 

The specific activities of the complex decline after reconstitution, as can be seen from Table 1. In the presence of an uncoupler a strong stimulation is observed, which indicates an efficient incorporation of the ATPase complex and a relatively low leakiness for protons. Thus, the ATPase proteoliposomes prepared from natural lipids function well at 50°C, the optimal growth temperature of SyneohooooQUS 6716. The specific hydrolysis and exchange activities are similar to those, found for reconstituted chloroplast ATPase (Pick, Racke2 1979). ... 

Pick U and Packer E (1979) Purification and reconstitution of the N-N -Dicyclo-hexylcarbodiimide sensitive ATPase complex from spinach chloroplasts, J. Biol. Chem. 254, 2793-2799. 

Proton ATPase complex was incubated with DMS (O) or DIG (A) after reconstitution into liposomes, at 0°G. At the time indicated in the figure, samples were taken to measure ATP-Pi exchange activity. The activity of the untreated complex was 60 n mol/mg protein/min. 

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

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... 

Studies with beef-heart submitochondrial particles initiated in Green s laboratory in the mid-1950s resulted in the demonstration of ubiquinone and of non-heme iron proteins as components of the electron-transport system, and the separation, characterisation and reconstitution of the four oxidoreductase complexes of the respiratory chain. In 1960 Racker and his associates succeeded in isolating an ATPase from submitochondrial particles and demonstrated that this ATPase, called F, could serve as a coupling factor capable of restoring oxidative phosphorylation to F,-depleted particles. These preparations subsequently played an important role in elucidating the role of the membrane in energy transduction between electron transport and ATP synthesis. 

The strong binding of the troponin T i region to tropomyosin should undoubtedly contribute to the steric stabilization of the position of the whole troponin complex on tropomyosin—actin. The Ca + sensitivity of actomyosin ATPase by troponin T2 is a little less cooperative than that by troponin T, and thus the depressive effect of free Mg " on the Ca sensitivity of the actomyosin ATPase with troponin T2 is less remarkable. Troponin T1 may be involved in these aspects. The maximum activation of actomyosin ATPase by troponin T2 in the presence of tropomyosin-troponin I-C was a litde less than that by troponin T. Troponin Ti itself depressed the ATPase and superprecipitation of actomyosin-tropomyo-sin-troponin I-C at all Ca + concentrations. The mixture of troponin Ti and T2 also depressed the ATPase at all Ca concentrations to the same extent as troponin Ti. This suggests that the native position of troponin Ti in the thin filament is different from that of isolated troponin T1 in the reconstituted filament. 

When the ATP synthase forms a part of the intact membrane system, it catalyzes proton translocation and ATP synthesis. However, when Fi is dissociated from Fq, the Fj no longer catalyzes ATP synthesis but rather ATP hydrolysis. Thus, Fi becomes an ATPase rather than an ATP synthase. Submitochondrial vesicles devoid of Fi can transport reducing equivalents, since they contain the redox carriers, but are unable to support ATP synthesis. Careful reconstitution of membrane vesicles by addition of Fi allows the complex to regain its capacity for ATP synthesis. 

A EXPERIMENTAL FIGURE 8-25 Mitochondrial particles are required for ATP synthesis, but not for electron transport. "Inside-out" membrane vesicles that lack Fi and retain the electron transport complexes are prepared as indicated. Although these can transfer electrons from NADFI to O2, they cannot synthesize ATR The subsequent addition of F-, particles reconstitutes the native membrane structure, restoring the capacity for ATP synthesis. When detached from the membrane, F-, particles exhibit ATPase activity. 

The interest aroused by the BLM has been mostly due to the fact that these systems form the basis for reconstitution of complex transport biomembrane systems, such as ionic channels, ATPases, the acetylcholine receptor, bacteriorhodopsins, etc. A first step in this direction was the discovery of a class of compounds that were found capable of radically affecting the electric... 

A number of active -subunits have been isolated from various Fi-ATPases after their complete dissociation into individual subunits (1-3). Two of these isolated / -subunits, the E, coli Fi P subunit(Ec 3) and the chloroplast CFi-/ subunit (CFi/ ), were shown to form hybrid FqFi-complexes upon their reconstitution into / -less R, rubrum chromatophores (3,4). These hybrid complexes restored a high Mg-ATPase activity, but very little ATP synthesis, in the inactive )5-less chromatophores. On the other hand, reconstitution of the native R, rubrum Fi-/3 subunit (Rr/3), that has been removed from the chromatophore membrane-bound FoFi-ATP synthase by extraction with LiCl (5,6) led to full restoration of both coupled ATP synthesis and hydrolysis by the / -less chromatophores. 

Extraction of thoroughly washed spinach thylakoids with 2M Li Cl in presence of Mg ATP, under the conditions developed for complete extraction of Ri/S from coupled R, rubrum chromatophores (5,6), led to 95% inhibition of their photophosphorylation activity. Fig. 1, lane 1 illustrates that in the 2M LiCl-extract, when compared with the same amount of CFi-ATPase protein (Fig. 1, lane 2), there is for each extracted CFifi also about 80% of CFxa and less than 30% of CF1-7, and c. This 2M LiCl-extract restored up to 30% of the photophosphorylation activity of the extracted thylakoids (10). The 2M LiCl-extract could reconstitute fi-less R. rubrum chromatophores and restored their Mg-ATPase activity as effectively as the native Rr/ (Table 1) and much more efficiently than the CFi/3 isolated from a dissociated CFi-ATPase (3). It should be emphasized that the CFi(- e) from which this GFi/3 ha been isolated did not bind to the 0-less chromatophores (3). It is therefore clear that, although the 2M LiCl-extract contains a mixture of CFi-subunits, it does not contain a fully assembled 03/ 37 complex. 

The observed single channel currents were carried by H through the isolated and reconstituted chloroplast ATPase. We demonstrate that it is the intact enzyme complex CFqCF] and not the membrane sector CFq alone that constitutes a voltagegated, proton selective channel with a hi unit conductance of 1-5 pS at pH 53-pH 8.0. The open probability P of the CFqCFj channel increased considerably with increasing membrane voltage (from Pq < 1% (V < 120 mV) to Pq < 30% (120 mV < V 200 mV)). In the presence of ADP (3 jxM) and Pj (5 jiM), wich specifically bind to CF the open probability decreased and venturiddin (1 i,M), a specific inhibitor of H flow through GFq in thylakoid membranes, blocked the chaimel almost completely. 

In the FoFi-ATPase of mammalian and yeast mitochondria, the oligomy-cin sensitivity conferring protein (OSCP) is one of the subunits of the enzyme complex, and is needed for inhibition of ATP hydrolysis by oligomycin (1). The amino acid sequence of OSCP from beef heart mitochondria is homologous to the amino acid sequence of the S-subunit of the Fi-ATPase from E. coli, 26.4% (2), chloroplasts, 25.3% (3), and Rhodospirill urn rubrum, 28.9% (2). However, only R. rubrum is sensitive to oligomycin (4). It has also been shown that R. rubrum Fj-ATPase, with the B-subunits substituted with B from E. coli, is not oligomycin sensitive when reconstituted to depleted membranes (5). 

Topographs of membrane proteins at subnanometer resolution were first acquired on highly ordered 2D reconstituted systems, that is, OmpF, bacteriorhodopsin (BR), water channels, potassium channels, halorhodopsin, outer membrane (OM) porins, adenosine triphosphate synthase (ATPase) " and light-harvesting (LH) complexes. " Target proteins were initially isolated from biological membranes and then reconstituted into lipid bilayers to form regular arrays. However, as described before, there is a certain... 

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