Quantification of rat sciatic nerve (Na+,K+)-ATPase by measurements of [3H]ouabain binding in intact nerve samples

The (Na+,K+)-ATPase concentration in rat sciatic nerve was quantified by measurements of [3H]ouabain binding to intact nerve samples in a vanadate-Tris buffer. This gave values of 145-172 pmol/g wet weight in mature female rats. The methodological errors of the [3H]ouabain binding assay was identified, quantified and corrected for. The [3H]ouabain binding sites were homogeneous with respect to [3H]ouabain affinity with an apparent dissociation constant of 5 X 10(-8) mol/l, i.e. in the same range as in samples of skeletal muscles incubated at similar conditions. Post-mortem the [3H]ouabain binding site capacity in rat sciatic nerve decreased with a half-life time of 5 days, allowing measurements to be performed within 6 h after death with a reduction in [3H]ouabain binding sites of only 3%. When nerve samples were frozen the [3H]ouabain binding site concentration remained constant. An increase in sciatic nerve [3H]ouabain binding site concentration of 122% was seen from the 2nd to the 4th week of life followed by a decrease of 53% to the 12th week of life. In mature males the [3H]ouabain binding site concentration was 1.8 times that in nerves from age-matched female rats. Denervation caused a reduction of 61%, whereas K+ depletion or thyroid status has no effect on sciatic nerve [3H]ouabain binding site concentration.