Structure of the Cation Occlusion Domain of Na+/K+ ATPase Studied by Proteolysis, Interaction with Ca2+ ions and Thermal Denaturation
Knowledge of the structure of cation binding sites and of organization of transmembrane segments of cation pumps, such as Na+/K+-ATPase, is essential for an understanding of the active transport mechanism. Renal Na+/K+-ATPase extensively digested with trypsin, in the presence of Rb+ and absence of Ca2+ ions, to a Cterminal 19kDa and smaller membrane-embedded fragments (8–11kDa) of the alpha chain, and intact beta chain or beta split into two fragments, is a valuable tool for establishing structure-function relationships. In these “19kDa membranes”, cation occlusion sites are intact, and although it is evident that the sites are located within trans-membrane segments, the identity and number of segments involved is unknown. The question as to which or how many of the trans-membrane segments actually occlude the ions is unanswered, and is the major focus of the present work. The first section of the present paper analyses the nature of the antagonism between Ca2+ and Rb+ ions in “19kDa-membranes”, and explains the previous observation that the presence of Ca2+ during digestion destabilises the 19kDa fragment to trypsin and occlusion is destroyed. The second section describes the purification and identification of the limit tryptic membrane-embedded fragments, produced by digestion of “19kDa-membranes” in the presence of Ca2+.
KeywordsHPLC Tyrosine Carboxyl Trypsin Stein
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