The H+, K+-ATPase of the gastric mucosa is the enzyme responsible for secretion of acid into the stomach. It is of considerable medical importance because disturbances in acid output appear to play a role in gastrointestinal diseases such as duodenal ulcer. Medical therapy for these diseases often involve treatments that alter acid secretion either by a direct inhibition of the pump or by influencing the regulation of its activity. Our long term objectives are to define the structural and functional domains of the gastric ATPase and to describe the number, sequence organization and regulation of its genes. During the past year, we isolated and characterized cDNAs containing the entire coding region of the rat stomach H+, K+- ATPase. Using the rat cDNA as a probe, we will isolate the gene(s) encoding the human gastric ATPase. The gene(s) will be characterized by restriction mapping and blot hybridization analysis and then the entire sequence will be determined. In addition to providing basic information about the structure of the gene, this study will provide cloned DNA from the human H+, K+- ATPase locus that could be useful in future studies as molecular probes in the analysis and diagnosis of disease states involving abnormalities in acid secretion. Using Northern blot and S1 analyses, we will examine the tissue distribution of the H+, K+- ATPase in the rat and analyze the levels of its expression during development. In order to study the structural and fucntional domains of the enzyme, we will develop expression systems which will be used to analyze the activity of the enzyme before and after modification of various domains. Full length cDNAs for the H+, K+-ATPase and the Na+, K+-ATPase will be used to construct a series of hybrid coding regions in vectors that allow expression in mammalian cells, in yeast or in an in vitro transcription/translation system. The high degree of sequence similarity between the two proteins and perfect amino acid alignment for almost 950 amino acids suggests that functional fusion proteins can be expressed. This will allow the identification and examination of the domains involved in cation specificity, energy transduction and the binding of drugs such as omeprazole. As information is acquired from these fusion protein experiments, a much finer structure/function analysis, at the level of individual amino acids, will be conducted using site- directed mutagenesis techniques.
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