H+-transporting ATPase

On the other hand, there are two groups or systems for various nutrients and metabolites in which transport of certain metabolites postulated to be driven directly by the hydrolysis of ATP or other energy-rich compounds without the requirement of a protonmotive force these are the binder -requiring systems and the phosphotransferase systems [22]. Binders are periplasmic proteins, which occur between the cytoplasmic and the outer membrane of microorganisms. They appear to be loosely attached to the outer face of the cytoplasmic membrane, from which they can be removed, for instance, by mild osmotic shock. However, the mentioned hypothesis that the (shock-sensitive) binder systems of active transport are primary active does not appear to be fully established mainly because a distinction between primary and secondary active transport is not possible in the presence of a functioning H -transporting ATPase. The main evidence in favor of primary active transport in shock-sensitive transport systems has been derived from a Ca, Mg, ATPase-less mutant of E. coli. There are, however, some findings which do not seem to support this hypothesis. 

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 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]. 

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... 

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. 

Gastric H,K-ATPase catalyses the 1 1 exchange of H for K upon hydrolysis of ATP, which results in vivo in a pH difference of more than six between the cytoplasm and the lumen [4-6]. Several reaction schemes have been developed to account for the hydrolysis of ATP and the transport of H and K" [14,104-107]. The basis for all these schemes is the Albers-Post scheme originally postulated for the mechanism of action of Na,K-ATPase [108,109]. 

Fig. 2. E]-E2 reaction cycle of H,K-ATPase, accounting for the transport of two H and two K ions per molecule of hydrolysed ATP. The ATPase reaction proceeds from 2H E, ATP through 2H E -P and 2H E2-P to 2K E. Details of the reaction cycle are described in the text.

Like Na,K-ATPase, gastric H,K-ATPase also exhibits a p-nitrophenylphosphatase (/>NPPase) activity. This phosphatase activity is dependent on Mg and K, or one of its congeners with the same order of selectivity as for the ATPase activity, yielding a specific activity of 6D84% of the maximal ATPase activity [4,136,137]. Phosphorylation by pNPP has not been demonstrated and transport is also not catalyzed by this substrate. As in the ATPase reaction the effect of on the... 

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]. 

J. M. Vanderkooi, A. Ierokomas, H. Nakamura, and A. Martonosi, Fluorescence energy transfer between Ca2+ transport ATPase molecule in artifical membranes, Biochemistry 16, 1262-1267 (1977). 

Omeprazole (p. 167) can cause maximal inhibition of HCl secretion. Given orally in gastric juice-resistant capsules, it reaches parietal cells via the blood. In the acidic milieu of the mucosa, an active metabolite is formed and binds covalently to the ATP-driven proton pump (H+/K+ ATPase) that transports H+ in exchange for IC into the gastric juice. Lansoprazole and pantoprazole produce analogous effects. The proton pump inhibitors are first-line drugs for the treatment of gastroesophageal reflux disease. 

This class of enzymes [EC 3.6.1.36] (also known as the hydrogen/potassium-exchanging ATPase, the potassium-transporting ATPase, proton pump, and the gastric H+/K+ ATPase) catalyzes the hydrolysis of ATP to ADP and orthophosphate, coupled with the exchange of and ions. The gastric mucosal enzyme has been the best characterized. 

The H+-K+-ATPase acts as the proton pump in the parietal cells of the stomach mucosa. It transports protons and Cl ions into the stomach via a K+ antiport. Substituted benzimidazoles, such as omeprazole (8.27) inhibit this enzyme and are 2-12 times as active as cimetidine (8.28) in inhibiting gastric stimulation. 

Proton pump inhibitors undergo rapid first-pass and systemic hepatic metabolism and have negligible renal clearance. Dose reduction is not needed for patients with renal insufficiency or mild to moderate liver disease but should be considered in patients with severe liver impairment. Although other proton pumps exist in the body, the H+,K+ ATPase appears to exist only in the parietal cell and is distinct structurally and functionally from other H+ -transporting enzymes. 

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