Quantitation of [3H]ouabain binding and turnover of Na-K-ATPase along the rabbit nephron.
To determine the number of Na-K-ATPase units and the enzyme's turnover rate along the rabbit nephron, the specific binding of [3H]ouabain and the Na-K-ATPase activity were measured in single nephron segments microdissected from collagenase-treated kidneys. The highest density of Na-K-ATPase (20-30 fmol X mm-1) was found in the distal convoluted tubule and the medullary thick ascending limb. Binding was intermediate (10 fmol X mm-1) in the proximal convoluted tubule and connecting tubule, and it was lowest (2-7 fmol X mm-1) in the pars recta, the cortical thick ascending limb, and the collecting tubule. In the medullary thick ascending limb, Scatchard analysis of the specific [3H]ouabain binding indicated a dissociation constant of 1.8 microM. The pump activity was proportional to the number of catalytic units, indicating that the maximal turnover rate of Na-K-ATPase (2,000 ATP molecules per minute per ouabain binding site) was similar in the various segments of the nephron. The method developed for quantitating [3H]ouabain binding is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine the number of Na-K-ATPase units in each region of the nephron.
D000818:Animals; D001665:Binding Sites; D002463:Cell Membrane Permeability; D007473:Ion Channels / Q000378:metabolism; D008297:Male; D009399:Nephrons / Q000201:enzymology / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014316:Tritium
D007473:Ion Channels; D014316:Tritium; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium