Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP.
Canine renal Na,K-ATPase was treated with ATP dialdehyde, "oxATP" (20 microM), as described by G. Ponzio, B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201-8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursor-product model to the data. Labeling with [3H]oxATP indicated about three sites per alpha beta protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the alpha and beta subunits and in small molecules at the tracker dye region. ATP (20 mM) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [3H]oxATP labeling. ATP also prevented [3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.
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Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000032:analysis; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000097:blood; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D000345:Affinity Labels; D008565:Membrane Proteins; D005557:Formaldehyde; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase