Urinary acidification is needed for normal acid-base homeostasis, and disorders of acidification are important causes of morbidity in chronic renal failure and other kidney diseases. My previous studies identified the characteristics of the proton pump responsible for urinary acidification, and showed that a major mechanism for regulation of acidification was changes in the number of pumps in the cell membrane brought about by rapid membrane insertion. The preliminary results on the isolation of a proton pump from the mammalian kidney are presented here. The goals of this proposal are to extend this work by completing the isolation of the renal proton pump using a combination of conventional column chromatography, and high performance liquid chromatography. The properties of the isolated pump will be examined to determine its overall structure, and to determine the contribution of changes in pump activity to the overall regulation of acidification. The isolated pump will be used to screen, by an enzyme-linked immunosorbent assay, for monoclonal antibodies specific to the proton pump. The antibodies will be characterized for affinity, and for the ability to detect differences and similarities between the kidney proton pump and other proton pumps. These antibodies will serve as probes for future studies on the synthesis and compartmentalization of the pump. These studies should enhance the understanding of how urinary acidification is regulated, and should give an insight into the abnormalities that may occur in renal disease.
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