ATPase preparations

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]. 

Soumarmon et al. [90,91] determined in an octylglycoside solubilized H,K-ATPase preparation by gel filtration and glycerol gradient centrifugation molecular masses between 390 and 420 kDa for the holomeric enzyme. 

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-... 

Essentially identical conclusions arose from the studies of Matthews et al. [138], An anti-peptide antibody directed against the cytoplasmically exposed C-terminal region of the Ca " -ATPase (985-994) reacted freely in native sarcoplasmic reticulum, in agreement with earlier observations [137], while the antibody directed against the putative luminal loop (877-888) reacted strongly only after solubilization of sarcoplasmic reticulum with Ci2Eg, Purified ATPase preparations reacted freely with both antibodies under both conditions. A 30-kDa protease-resistant fragment obtained... 

The possible role of cellular glutathione status in the controlling sarcolemmal protein activity has been addressed by studying the effect of GSH, GSSG and several other thiol and disulphide compounds on Na/K ATPase activity using (1) an isolated bovine ventricular Na/K ATPase preparation (2) crude sarcolemmal preparations (biochemical studies) (c) Langendorff-perfiised isolated hearts (cytochemical studies) and (4) isolated ventricular myocytes (electrophysiologjcal studies). 

Although it is widely accepted that ischaemia/ reperfusion-induced oxidant stress is associated with a reduction of Na/K ATPase activity, it is difficult to determine which features of this process are responsible for this effect. A classical approach to this type of problem has been to determine the effect of the application of selected metabolites or agents on the activity of the enzyme of interest, an approach that has been exploited for the sarcolemmal Na/K ATPase and glutathione (Haddock et al., 1990). The application of GSH (O.l-l.OmM) induces a concentration-dependent increase in the activity of a bovine isolated ventricular Na/K ATPase preparation (determined by the ouabain-sensitive hydrolysis of ATP to release inorganic phosphate). In the presence of 1 mM GSH there was a 38% stimulation of activity compared to untreated control... 

Figure 4.9 Effect of reduced glutathione (GSH) (0.25-1.0 ihm) and oxidized glutathione (GSSG) (0.25-1.0 mM) on ouabain-sensitive Na/K ATPase activity. An isolated Na/K ATPase preparation was prepared from fresh bovine ventricular tissue. Na/K ATPase activity was determined and quantified by the ouabain-sensitive hydrolysis of ATP to yield Inorganic phosphate. The rate of inorganic phosphate production was compared prior to and following the addition of either GSH or GSSG to the Incubation mixture. The data are presented as...

The effect of gastric HVK -ATPase inhibitors on enzyme activity (ATP cleavage) can be studied in vitro with partly purified HVK -ATPase preparations [27]. This assay has been used more effectively to study the mechanism of action of H /K -ATPase inhibitors in detail than to study the structure-activity relationship of such inhibitors [28]. Since HVK -ATPase inhibitors of the omeprazole-type need acid activation and the enzyme assay should be performed at neutral pH values, a pre-incubation period at the lowest possible pH of about 6 was used to initiate the acidic conversion of the test compound into its active principle. This reflects more the chemical instability of the test compound at neutral pH values than its effect during conditions of much higher acidity within the secretory cannaliculus of the parietal cell during acid secretion. Many chemically labile inhibitors are therefore very active in this test system. However, they do not cause an inhibition in more complex test systems and, therefore, are without any practical usefulness [28]. 

The effect of gastric PPIs on ATPase activity (ATP-induced phosphorylation) can be studied in vitro with partially purified H /K -ATPase preparations Ifom pig gastric mucosa (36). Given that the enzyme assay needs to be performed at neutral pH, this system has been most effectively used to study the mechanism of action of PPIs rather thmi the structure-activity relationship (SAR)... 

To test the tissue specificity of these observations the effect of the activating ions was surveyed using Na-K ATPase prepared from calf brain... 

Na-K ATPase preparation was incorporated into phosphatidyl choline vesicles containing radioactive Na and [129]. Upon incubation of the vesicles, half of which were inside out, and thus could respond to external ATP, in a medium containing ATP, Mg +, Na" and, uptake of 3 mol Na and extrusion of 2 mol per mol ATP hydrolyzed was observed. Transport and ATPase activity were more than 90% inhibited by intravesicular ouabain, thus at the side opposite to the ATP-activation side. 

Upon purification, the K -stimulated phosphatase activity is always copurified with the (K )-ATPase activity [63-65]. Mitochondrial markers, such as cytochrome c oxidase, succinate dehydrogenase, monoamino-oxidase, and the ribo-somal marker RNA are largely removed by the purification procedure. The same is true for the anion-sensitive ATPase and 5 nucleotidase activities, but some (Na — K )-ATPase activity is still present in highly purified (K" -I-H )-ATPase preparations. Purification is also characterised by a lowering of the K -insensitive Mg ATPase activity, but even in the purest preparations some Mg -ATPase activity (4% of (K -I- H )-ATPase activity) is still present. This may represent an impurity or an inherent property of the enzyme. 

Electronmicroscopic studies show that the purified ATPase preparation consists almost entirely of smooth-surfaced vesicles with a diameter of 0.1-0.2 jam [63,66,67] and a volume of 2jal/mg protein [67,68]. The general organisation of in-tramembranous particles seen after freeze-fracture suggests that the vesicles may originate from the tubulovesicular system of the intact parietal cells [67]. This system is abundant in the apical region of these cells and appears to be directly involved in the acid secretion process [69]. 

The membrane preparation, which is enriched in (K + H )-ATPase, contains in addition to proteins a considerable amount of cholesterol and phospholipids. The content and composition of this lipid fraction (high cholesterol and sphingomyelin content, Table 4) is characteristic for a plasma membrane fraction as is also found for a purified (Na + K )-ATPase preparation [1]. 

Saccomani et al. [85] treated a vesicular (K + H )-ATPase preparation from pig gastric mucosa with phospholipase Aj, resulting in a breakdown of 50% of the phospholipids. This treatment also results in partial loss of ATPase activity, but the residual activity is still 25% of the original activity. The K -stimulated phosphatase activity is not affected by this treatment. Schrijen et al. [86] used two phospholipases with shght difference in substrate specificity, alone and in combination. With each of these phospholipases approx. 50% of the phospholipids could be hydrolysed resulting in a 50% loss in enzyme activity. When the two phospholipases were used successively 70% of the phospholipids were hydrolysed and the loss of activity was also 70%. This represents a striking parallelism between residual phospholipid content and ATPase activity. In this case the K -stimulated phosphatase activity... 

Effect of phospholipase treatment on phospholipid composition and activity of (K +H )-ATPase preparation... 

Membrane enzymes often show a transition in the Arrhenius plot of the enzyme activity, which is usually attributed to a change in the lipid configuration. (K" " -h H" ")-ATPase preparations show such a transition at 27-28°C [71,86]. However, a transition in the polarisation signal of a lipid viscosity probe (diphenylhexatriene), was not observed in this region [71], indicating that the transition in the ATPase activity is either due to a change in the rate-limiting step of the reaction or to the presence of an annulus of specific lipids with physical properties different from those of the bulk lipids. 

Approach to the chemotopographical difference between the digitalis binding matrix of Na /K -ATPase preparations from human cardiac muscle and human brain cortex Conclusions... 

As shown elsewhere [78], the determination of structure-activity relationships and receptor kinetics, extrapolatable to man, requires the use of Na /K -ATPase preparations from human tissues. Autopsy preparations are perfectly suitable when taken within 12 hours post-mortem [78,79]. The enzyme should be only partially purified to avoid the various possible artefacts of intensive purification procedures [80-82]. The interaction of cardiac glycosides and NaYK -ATPase is controlled by Na, Mg, and... 

Table 4.1. IMPACT OF CHANGES IN THE GEOMETRY OF CID-cis STEROIDS OF THE DIGITALIS TYPE ON THE APPARENT GIBBS ENERGY OF INTERACTION (-AG ) WITH NaYK -ATPase PREPARATIONS FROM HUMAN HEART (HH), HUMAN BRAIN CORTEX (HB), AND GUINEA-PIG HEART (GH) REPERCUSSIONS ON THE ENERGETIC CONTRIBUTIONS OF THE SIDE-CHAINS AT C-3fi AND C-np. METHODOLOGICAL DETAILS IN [87,88].

Hydroxyl groups at the steroid skeleton are known to determine the type of hormonal action. Thus, the impact of hydroxyl groups in cardiac steroid action demands some consideration. Disappointingly, hydroxylation always reduces the energy of interaction of digitalis compounds with the Na /K -ATPase preparations from human cardiac muscle and brain cortex as well as from guinea-pig cardiac muscle as numerically differentiated in the following, based on data in Table 4.4. 

APPROACH TO THE CHEMOTOPOLOGICAL DIFFERENCE BETWEEN THE DIGITALIS BINDING MATRIX OF NaYK -ATPase PREPARATIONS FROM HUMAN CARDIAC MUSCLE AND HUMAN BRAIN CORTEX... 

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