Gastric vesicle, isolated

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. 

Lorentzon P, Jackson R, Wallmark B, et al. Inhibition of (H + K )-ATPase by omeprazole in isolated gastric vesicles requires proton transport. Biochim Biophys Acta 1987 897 41-51. 

One of our substituted benzimidazoles was, however, tested on isolated vesicles containing the acid pump enzyme in George Sachs s laboratory. To our disappointment, no effect was observed. Meanwhile, Bjorn Wall-mark joined the project with the aim of studying the effects of substituted benzimidazole compounds on the acid pump mechanism. He introduced the technique with isolated gastric vesicles at the Astra Hassle biochemical laboratory and confirmed Sachs s negative results. We then decided to do more basic research on the HVK -ATPase, including characterization of... 

Figure 9. pH dependency for inhibition of H /K -ATPase by H 168/68 (omeprazole) in isolated gastric vesicles from rabbit. Adopted from ref. 24. 

Hall K, Perez G, Mayer P, Gutierrez C, Munson K, Hersey, SJ, Kaplan JH, Sachs G (1990) Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa. Biochemistry 29 701-706... 

Preparation of H,K-ATPase enriched microsomal vesicles. H,K-ATPase-containing gastric microsomal vesicles were isolated from rabbit stomach as previously described (12). Crude microsomes were harvested from homogenized mucosa of unstimulated rabbit stomach (H2 receptor-blocked) as the membrane pellet sedimenting between 10 min at 13,000 x g and 1 hr at 100,000 x g. The pellet was resuspended in 10% sucrose, brought to 40% sucrose (9 ml), and overlaid with successive layers of 30% sucrose (11 ml), 10% sucrose (16 ml) [300 mM sucrose, 5 mM tris(hydroxymethyl)aminomethane (Tris), and 0.2 mM EDTA, pH 7.4] in a 37 ml tube. After centrifugation at 80,000 x g for 4 hr, the purified gastric microsomal vesicles were collected from the interface between 10% and 30 % sucrose and stored at 4° C until use. 

The relative potency of the various substituted benzimidazoles was identical in vivo (dog) and in vitro (isolated guinea pig gastric mucosa, gastric glands and isolated vesicles) [27]. Kinetic studies of the inhibition in isolated human glands confirmed the noncompetitive type of inhibition found in isolated guinea pig gastric mucosal preparation. 

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