ATPases, membrane embedded

The results of all of these topography studies are summarized in Fig. 1. The open eircles indicate residues of the H -ATPase shown in one way or another to be located on the cytoplasmic side of the membrane and the closed circles indicate residues in membrane-embedded segments. The lines in the sequence indicate minor regions with locations as yet not established. Thus, the topographical locations of nearly all of the 919 residues in the molecule have been established. It should be emphasized that the exact points of entry and exit of the polypeptide chain into and out of the membrane are not implied in the model. 

With this information in hand, we may now consider how the -ATPase polypeptide chain might fold into its functional three-dimensional structure. First, regarding the actual number of membrane-spanning stretches, the available experimental data indicate only that each of the three membrane-embedded peptides must have an even number of and a minimum of two such stretches. However, hydropathy analysis by the method of Mohana Rao and Argos [48] suggests that the second mem-... 

Except for the two additional membrane-spanning stretches in the second membrane-embedded segment, the proposed membrane-embedded stretches indicated in Fig. 1 are similar to those proposed for the closely related Ca -ATPase [53]. In fact, the overall topography proposed in Fig. 1 is quite similar to the Ca -ATPase model, lending additional credibility to each. 

Second, our studies of the secondary structure of the H" "-ATPase indicate that about 36% of the polypeptide chain is present in a helical configuration [27,42]. If the membrane-embedded sector of the molecule is helical as shown, only 90 or so additional residues in the molecule can be present as helices. Thus, the great majority... 

Further considerations here do not depend critically on the accuracy of the working model of Fig. 2. Indeed, the interdomain cleft may as well be at the side of the molecule near the surface of the membrane as can be imagined from inspection of the structures proposed by Taylor et al. [75] and Stokes and Green [76] for the Ca -ATPase. It is only important to stipulate that the molecule contains at least two domains and a cluster of membrane-embedded helices. 

ABC transporters are involved in both uptake and excretion of a variety of substrates from ions to macromolecules. Whereas export systems of this type are present in all kingdoms of life, import systems are exclusively found in prokaryotes. ABC transporters are minimally composed of two hydrophobic membrane embedded components and two ATPase units. 

In parallel, activity of adenylate cyclase is inhibited by Ca2+, and the ion activates the membrane-embedded CAM to close the cAMP-dependent channels. In addition, CAM-PDE is induced to hydrolyze the messenger molecules rapidly. 4) Finally, Ca2+ activates the cytoplasmic CAM to enhance the activity of Ca2+-translocating ATPase, causing the cells to return to the resting state. 

A membrane-embedded hairpin structure for the c subunit was established from genetic studies.31 The role of cAsp-61 in the second membrane helix of the c subunit has been studied in detail the cAsp-61— Asn or Gly mutants showed no proton conduction or ATP synthesis.99 Binding of DCCD to this residue blocked proton conduction. These results are consistent with the notion that cAsp-61 is part of the proton pathway. The carboxyl residue at position 61 was able to be transferred to the corresponding position of the first helix.100 Vacuolar-type ATPase has a similar proteolipid subunit possibly evolved from the same ancestral protein as the c subunit.25 The vacuolar proteolipid has glutamate in the middle of the fourth membrane-spanning helix, corresponding to the position of c Asp-61 of the c subunit. The glutamate may play an important role in proton conduction similar to the c subunit.101 ... 

The lack of effect of steroids on the ATPase activity in the assay with Commel ina protoplasts is no proof against an interaction with this membrane protein as the experiment has been performed in the presence of a detergent (Triton X-100), that is, under conditions where the protein is not capable of vectorial transport. This result is, rather, a hint for an interaction with the membrane-embedded moiety of the proton pump. 

Nevertheless, it was clearly demonstrated that bras-sinosteroids, like the other steroids investigated here, interact with the plasmalemma thereby showing short-term effects on the membrane potential and/or medium acidification. In some cases these effects correlate with stomata 1 movement and solute uptake into leaves or conducting tissue. The results are compatible with an effect of these substances on the membrane-embedded moiety of the plasmalemma H -ATPase yielding a modified proton pump rate. They support, therefore, the "Annulus Hypothesis" according to which sterol effects are caused by direct lipid-protein binding. 

Figure 5.3 Na —K —ATPase pump embedded in a cell membrane.

It is easy to extend this model in order to interpret any kind of experimental data on multiple reiterative ATP synthesis initiated by the creation of an artificial transmembrane difference of the electrical potentials, A<, generated across the closed vesicles, e.g., liposomes encrusted with membrane-embedded ATPases. In this case, when the reiterative acts of ATP formation are driven by Aq> in the lack of ApH, the protonation of the ionized acid group A is evidently provided by the field-induced decrease in the pK value of the ionizable group AH (Fig. 5.26). The Acp difference imposed across the coupling membrane can also lead to the occurrence of the difference in the proton concentrations related to the local regions in the vicinities of gates a and b. This difference, induced by the electric field, can exist even without any pH difference between the bulk phases on both sides of the coupling membrane. 

The procedure for purification of Na,K-ATPase in membrane-bound form from the outer renal medulla of mammalian kidney offers the opportunity of exploring the structure of the Na,K-pump proteins in their native membrane environment. The protein remains embedded in the membrane bilayer throughout the purification procedure thus maintaining the asymmetric orientation of the protein in the baso-lateral membrane of the kidney cell in the purified preparation. This preparation has been particularly useful in studies of ultrastructure, protein conformation and for... 

F-ATPases (including the H+- or Na+-translocating subfamilies F-type, V-type and A-type ATPase) are found in eukaryotic mitochondria and chloroplasts, in bacteria and in Archaea. As multi-subunit complexes with three to 13 dissimilar subunits, they are embedded in the membrane and involved in primary energy conversion. Although extensively studied at the molecular level, the F-ATPases will not be discussed here in detail, since their main function is not the uptake of nutrients but the synthesis of ATP ( ATP synthase ) [127-130]. For example, synthesis of ATP is mediated by bacterial F-type ATPases when protons flow through the complex down the proton electrochemical gradient. Operating in the opposite direction, the ATPases pump 3 4 H+ and/or 3Na+ out of the cell per ATP hydrolysed. 

The question of whether an enzyme is membrane bound or membrane associated is to some extent a matter of semantics. However, it is certainly true that some proteins are readily dissociated from membranes whereas others require quite drastic conditions before they can be dissociated from the membrane. As limiting cases, the former can be designated as membrane associated and the latter as membrane bound. Enzymes that are generally considered membrane bound are firmly embedded in the membrane structure. For example, the mitochondrial coupling factor is strongly coupled to the bilayer structure by hydrophobic polypeptides. The Na+-K+ ATPases that have been purified have a small patch of associated phospholipids when the enzyme is delipidated, enzymatic activity is lost. In fact, membrane-bound enzymes appear to be... 

The orientation of the j8-polypeptide has been explored by the use of lipophilic affinity labelling reagents generated photochemically inside the membrane. This shows the region of the polypeptide embedded in the lipid bilayer. The probe 0-hexanoyl-3,5-diiodo-JV- 4-azido-2-nitro-phenyl)tyramine undergoes photochemical conversion into the reactive nitrene, and has been used to label the (Na+, K+)-ATPase from Bufo marinus toad kidney. This shows that the j8-polypeptide is also a transmembranous polypeptide,49 a view that is in accord with immunochemical evidence.50... 

The outer membrane, the plasmalemma, efficiently protects the cell from the environment while, at the same time, carrying out functions important for cell metabolism the uptake of substrates and the elimination of toxic compounds. Substrate exchange with the environment is controlled by transport proteins embedded in the membrane (energy-requiring pumps such as Na+,K+-ATPase, or other transport units such as the Na+/glucose cotransporter and sodium and calcium ion channels) [1],... 

These results are in agreement with the aforementioned findings and support the assumption that drug-membrane interactions leading to severe changes in membrane structure (domain formation and heterogeneity) can affect the functioning of embedded proteins such as Na+, k+-ATPase, P-gp, and PKC. 

The mechanics of converting proton flow back into mitochondria into high-energy phosphate bonds is performed by the FoFj ATPase. This complex enzyme system spans the inner mitochondrial membrane and appears as particles labeled EP in Figure 17.3. Structural details are given in Figure 17.7. The enzyme consists of two sections the F0 section is embedded in the membrane and forms a channel through which protons are permitted to enter the F, section. The latter is located on the matrix side of the membrane and is attached to the F0 section. The ATPase catalyzes the formation of ATP from ADP and P,. The reverse reaction, ATP —> ADP + P, is carried out when Fj is separated from F0 hence the term ATPase. 

Figure 17.7 FqFj ATPase. The F0 section is embedded in the inner mitochondrial membrane, whereas Fj section is located on the matrix side. The various subunits of Fj are designated as a, (3, y, 8, and e.

Na+K+ ATPases, so-called sodium-potassium pumps (Figure 4.6), embedded in the basolat-eral membrane. The sodium-potassium pump is a highly conserved integral membrane protein, expressed in virtually all animal cells. The transport of sodium creates both an electrical and a chemical gradient across the plasma membrane. In turn this provides ... 

Of the two mitochondrial membranes (Fig. 1-9), the outer one is more permeable to various small anions and cations (e.g., H+), adenine nucleotides, and many other solutes than is the inner one. The inner membrane invaginates to form the mitochondrial cristae, in which the enzymes responsible for electron transfer and the accompanying ATP formation are embedded. For instance, the inner membrane system has various dehydrogenases, an ATPase, and cytochromes (discussed in Chapters 5 and 6). These proteins with enzymatic activity occur in a globular form, and they can be an integral part of the membrane (Fig. 1-8) or loosely bound... 

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