H,K ATPase a subunit

In the case of the Na,K ATPase, the last 161 amino adds of the a subunit are essential for effective assodation with the 6 subunit. Further, the last four or five C-terminal hydrophobic amino adds of the Na -pump 6 subunit are essential for interaction with the a subunit, whereas the last few hydrophilic amino adds are not. Expression of the Na -pump a subunit, along with the 6 subunit of either the sodium or proton pump in Xenopus oocytes, has shown that the B subunit of the gastric proton pump can act as a surrogate for the 6 subunit of the sodium pump for membrane targeting and Rb uptake. This implies homology in the associative domains of the 6 subunits of the two pumps. The H,K ATPase a subunit requires its a subunit for effident cell-surface expression. Expression of chimers of the a subunits of the Na,K and Ca ATPases showed that the C-terminal half of the a subunit assembled with the B subunit. 

Gedda K, Scott D, Besancon M et al (1995) Turnover of the gastric H, K" -ATPase a subunit and its effect on inhibition of gastric acid secretion. Gastroenterology 109 1134-1141... 

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]. 

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. 

The recent identification of the H,K-ATPase P subunit and the demonstration that the ratio for the H,K-ATPase catalytic a subunit/) subunit is about 1 1 [62,71] might bring new insight into the structural and functional assembly of H,K-ATPase in situ. 

Horisberger JD, Jaunin P, Reuben MA, Lasater LS, Chow DC, Forte JG, Sachs G, Rossier BC, Geering K (1991) The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps. J Biol Chem 266 19131-19134... 

Shin JM, Sachs G (1996) Identification of a region of the H,K ATPase alpha subunit associated with the beta subunit. J Biol Chem 269 8642-8646... 

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. 

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. 

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. 

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