H-ATPase

The mitochondrial ATPase system functions as a proton translocating, reversible ATPase and is thus the proximate cause for ATP synthe- 

Studies employing mitochondrial ATPase reconstituted into pro-teoliposomes and evaluating its ability to create a membrane potential with the penetrating ion method have shown clearly that the ATPase can generate a membrane potential. Drachev et al. (1976) showed that when ATP was added to the reconstituted ATPase lipsome in the presence of Ca , a transmembranous electric potential difference was detected. 

By appropriate treatment of the ATPase and its reconstitution into liposomes, it is possible to show that the H+-ion translocation and ATP-hydrolyzing activities of this complex are separable. Thus, coupling factor Fi, devoid of hydrophobic proteins, is able to carry out hydrolysis of ATP, whereas the addition of hydrophobic proteins to the ATPase increases transport, such transport being sensitive to inhibition by oligomycin. These studies have been reviewed by Skulachev (1975). 

Electron transport and oxidative phosphorylation represent the most complex membrane processes yet uncovered in living mammalian cells. The broad outlines of this pathway are not much in question. Thus, the oxidation of one molecule of NADH by the respiratory chain results in the formation of three molecules of ATP. Three complexes along this chain—NADH-Q reductase, QH2-cytochrome c reductase, and cytochrome c oxidase—contain the sites where energy is transduced and enabled to interact with the ATPase to generate ATP (Fig. 2). Abundant evidence exists, as well, for the transfer of energy among these three complexes without the involvement of ATP synthesis for instance in ion translocation by the mitochondrion (Emster, 1977). 

The manner in which the energy liberated during the movement of electrons down the respiratory chain is coupled to the formation of ATP has been the subject of elaborate theorizing and investigation, often not without its acrimony. The early history of this field has been captured in a collection of papers edited by Kalckar (1969). Recently, the Annual Review of Biochemistry (Boyer et al., 1977) published six thoughtful essays written by leaders in this field which were put together in a spirit of compromise and further experimentation. 

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FIGURE 10.13 Some of the sequence homologies in the nucleotide binding and phosphorylation domains of Na, K -ATPase, Ca -ATPase, and gastric H, K -ATPase. (Adapted from j0rgensm, P. L., and Andersen, J. R, 1988. Structnral basis for Ei - E2 confoyinational transitions in Na, K -pnmp and Cc -pnmp proteins. Journal of Membrane Biology 103 95-120)... 

FIGURE 10.16 The H+,lO-ATPase of gastric mucosal cells mediates proton transport into the stomach. Potassimn ions are recycled by means of an associated K /Cl cotransport system. The action of these two pnmps results in net transport of and Cl into the stomach. 

The discovery of the antiulcer activity of H2 antihistamine antagonists has revolutionized the treatment of that disease. A benzimidazole. Omeprazole (55), inhibits gastric secretion and subsequent ulcer formation by a quite different mechanism. Studies at the molecular level suggest that this compound inhibits K /H dependent ATPase and consequently shuts down the proton pumping action of this enzyme system. 

The H,K-ATPase, expressed in the parietal cells of the stomach, transports H+ ion from cytoplasm to lumen in exchange for extracytoplasmic K+ ion in an electroneutral exchange using the energy of ATP hydrolysis. 

Tlie Na+/K+-ATPase belongs to the P-type ATPases, a family of more than 50 enzymes that also includes the Ca2+-ATPase of the sarcoplasmic reticulum or the gastric H+/K+-ATPase. P-Type ATPases have in common that during ion transport an aspartyl phos-phointermediate is formed by transfer of the y-phosphate group of ATP to the highly conserved sequence DKTGS/T [1]. 

Gastric H,K-ATPase inhibitors Potassium competitive acid blockers... 

The proton pump is the gastric H,K-ATPase, which secretes hydronium ions, H30+, in exchange forK+ into the secretory canaliculus generating a pH of <1.0 in... 

Since a substituted benzimidazole was first reported to inhibit the H,K-ATPase by covalent binding [ 1 ], many PPIs have been synthesized and are in clinical use. These all have a similar core structure, 2-pyridy lmethylsulfinyl benzimidazole moiety except tenatoprazole. Tenato-prazole has 2-pyridylmethylsulfinyl pyridoimidazole moiety. 

Proton Pump Inhibitors and Acid Pump Antagonists. Figure 2 Chemical mechanism of irreversible PPIs. PPIs are accumulated in acidic lumen and converted to active sulfenic acid and/or sulfenamide by acid catalysis. These active forms bind to extracytoplasmic cysteines of the gastric H.K-ATPase [3]. 

Fellenius E, Berglindh T, Sachs Getal(1981) Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+)ATPase. Nature 290 159-161... 

Shin JM, Cho YM, Sachs G (2004) Chemistry of covalent inhibition of the gastric (H+,K+)-ATPase by proton pump inhibitors. J Am Chem Soc 126 7800-7811... 

Shin JM, S achs G (2004) Differences in binding properties of two proton pump inhibitors on the gastric H+,K+-ATPase in vivo. Biochem Pharmacol 68 2117-2127... 

Fig. 2b. The appearance of two crystal forms shows that the protein in the membrane exists in equilibrium between the protomeric aj8 unit and oligomeric (aj8>2 forms. The high rate of crystal formation of the protein in vanadate solution shows that transition to the E2 form reduces the difference in free energy required for self association of the protein. This vanadate-method for crystallization has been very reproducible [34-36] and it also leads to crystalline arrays of Ca-ATPase in sarcoplasmic reticulum [37] and H,K-ATPase from stomach mucosa [38]. 

In the family of cation pumps, only the Na,K-ATPase and H,K-ATPase possess a p subunit glycoprotein (Table II), while the Ca-ATPase and H-ATPase only consist of an a subunit with close to 1 000 amino acid residues. It is tempting to propose that the p subunit should be involved in binding and transport of potassium, but the functional domains related to catalysis in Na,K-ATPase seem to be contributed exclusively by the a subunit. The functional role of the P subunit is related to biosynthesis, intracellular transport and cell-cell contacts. The P subunit is required for assembly of the aj8 unit in the endoplasmic reticulum [20]. Association with a j8 subunit is required for maturation of the a subunit and for intracellular transport of the xP unit to the plasma membrane. In the jSl-subunit isoform, three disulphide... 

The P subunit is not as well conserved as the a subunit, with 91 % overall homology between the P subunit of sheep, pig, and human and 61% between the p subunit of human and Torpedo. Homologies between -subunit isoforms and the p subunit of H,K-ATPase are moderate, 25-30%, but some segments are well conserved. As shown in Table II, beta signatures 1 and 2 are preserved in the (6-subunit isoforms and P subunit of H,K-ATPase [70] as an indication that the positions of tryptophans and cysteines are well conserved elements. 

A number of reviews dealing with the physiological role of H,K-ATPase in acid... 

In this chapter we will review the recent investigations of the structure of both the a and P subunit, and the function of gastric H,K-ATPase. We will proceed from a brief overview of the tissue distribution to a successive discussion of structure, kinetics, transport properties, lipid dependency, solubilization and reconstitution, and inhibitors of H,K-ATPase that may label functionally important domains of the enzyme. 

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

Until recently, the possibility that H,K-ATPase consists not only of a catalytic a subunit but also of other subunits was not examined. This was mainly due to the fact that SDS-PAGE of purified gastric H,K-ATPase preparations principally gave one protein band with an apparent molecular mass of about 100 kDa, which was reported to comprise 75% or more of the total amount of protein [6,66,67]. This mass is lower than the mass deduced from its cloned cDNA [40], but may be due to the higher electrophoretic mobility of membrane-bound proteins, as consequence of having relatively high contents of hydrophobic amino acid residues [68]. 

One of the first observations suggesting that the functional unit of H,K-ATPase is not a monomer of the catalytic subunit, but instead a heterodimer of the catalytic... 

Further indications for an additional subunit were provided by a crosslinking analysis of C Eg solubilized H,K-ATPase, which exhibited ATPase and phosphatase activities, and ligand affinities comparable to the native enzyme [70]. Glutar-aldehyde treatment of soluble protein fractions resolved on a linear glycerol gradient revealed no active fraction enriched in monomeric (A/p = 94 kDa) H,K-ATPase. Instead, K -ATPase activity was only obtained in fractions enriched in particles of Mr = 175 kDa. This size also suggested that the functional H,K-ATPase unit is a heterodimer of a catalytic subunit and an additional subunit, since the apparent molecular mass of 175 kDa is probably too small to be a homodimer of the catalytic subunit. 

Hall et al. [62] identified in a separate study the same glycoprotein in H,K-ATPase vesicles isolated from porcine gastric mucosa. A stoichiometric ratio of 1.2 1.0 was found for the deglycosylated protein (35 kDa)/catalytic 94-kDa protein. Furthermore, compelling evidence that this glycoprotein is the H,K-ATPase p subunit was provided by N-terminal sequence analysis of three protease V8-obtained peptides of the 35-kDa core protein. These peptides showed 30% and 45% homology with the Na,K-ATPase pi and pi subunit, respectively. 

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

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