Investigation of reaction mechanism and higher oligomeric structure of Na-pump based on single-molecule observation technique.

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(1) Protomeric images of Na/K-ATPase and H/K-ATPase were observed by electron microsoopy(EM) after rotary-shadowing the solubilized enzyme preparation. Without cross-linking the preparation, dimeric and tetrameric molecules were detected by EM, indicating that the pump molecules are assembled in oligomer(2) We have established the method for determining the oligomericity of P-type ATPases by means of single-molecule observation of fluorescently labeled enzyme using the total internal reflection fluorescence microscopy.(3) Tetrameric structure of P-type ATPases has been determined not only Na/K-ATPase but also H/K-ATPase. In case ofH/K-ATPase, functional unit of the enzyme was shown to be tetraprotomer based on the relationship between the enzyme activity and the oligomeric structure.(4) Analysis of the single mutations ofATP-binding region of Na/K-ATPase suggested that the ATPase molecule has one ATP binding site, which changes the affinity for ATP depending on the ligands present and the ATPase molecules form oligomer to maintain the interaction between them. We have estimated the third Na+ binding region of Na/K-ATPase at the first time.