Α-subunit Of Na Research Articles

Characterization of the membrane-bound ATPase from a facultatively anaerobic alkalophile

We have studied the properties of membrane-bound ATPase of a facultatively anaerobic alkalophile. The enzyme could not be solubilized without detergent, suggesting an integral membrane protein. The activity was accelerated by NH 4 + and acetate anion, and inhibited by NO 3 −. The enzyme required Mg 2+ or Mn 2+ as a divalent cation for the maximal activity. In addition to ATP, the enzyme utilized other triphosphates of nucleosides as a substrate, but not di- nor monophosphates. The enzyme was suggested to crossreact with an antibody against the α-subunit of Na +/K +-ATPase from dog kidney.

Immunohistochemical localization of Na+,K+-ATPase in human liver.

The localization of Na+, K+-ATPase in human liver was investigated on the light and electron microscopic levels by using anti-sera on the catalytic subunit of Na+, K+-ATPase in human kidney. α-subunit of Na+, K+-ATPase was extracted from the gel of SDS-PAGE as an immune antigen and injected into rabbits in order to produce the antibody. Under light microscopic examination, reaction products were recognized at the bile canalicular, lateral and sinusoidal membranes of hepatocytes and luminal membrane of biliary epithelial cells on the PLP (periodate-lysine-paraformaldehyde) fixed frozen sections. By ultrastructural observation, reaction products were also recognized at the bile canalicular membrane of hepatocytes and luminal membrane of biliary epithelial cells. These results suggest that Na+, K+-ATPase is responsible for bile formation and transport processes on these membranes.

High-affinity 86Rb-binding and structural changes in the α-subunit of Na +,K + -atpase as detected by tryptic digestion and fluorescence analysis

High-affinity 86Rb-binding has been related to tryptic cleavage and fluorescence from intrinsic and extrinsic probes in order to examine the relationship of cation binding to structural transitions in the α-subunit of pure membrane-bound Na +,K + -ATPase from the outer renal medulla. Native Na +,K + -ATPase binds two Rb + ions per α-subunit (12.3 nmol/mg protein) with high affinity ( K d = 7.5 μM) in 25 mM Tris-HCI, pH 7.5. Enzyme with one molecule of covalently attached fluorescein per γ-subunit has the same capacity (12.8 nmol/mg protein) but a much lower affinity for Rb + ( K d = 29.2 μ M). The changes in conformational state of the protein are correlated with occupancy of the high-affinity sites for Rb +, also at concentrations of Rb + below the K d . Titration at varying ionic strength suggests that the E 2-form is the relaxed or native conformation of the α-subunit. Changes in tryptic digestion pattern and in fluorescence are parallel events both in the conditions of the binding assay and at physiological ionic strength. Reversible blocking of sulfhydryl groups with Thimerosal (ethylmercurythiosalicylate) abolishes the fluoresence responses to K + or Rb + withont affecting the capacity or the affinity for binding of 86Rb. The demonstration of high-affinity binding of Rb + without coupling to a conformational change suggests that the E 1-form of the protein exposes sites for tight binding of K + or Rb + at the cytoplasmic membrane surface.