Studies on ouabain-binding to (Na + + K +)-ATPase from Malpighian tubules of the locust, Locusta migratoria L.

Abstract

A study has been made on the binding of [ 3H]ouabain to (Na + + K +)-ATPase in microsomal preparations from Malpighian tubules of Locusta migratoria. The rate constants at 30°C were 1.5 · 10 3 ± 3.5 · 10 2 M −1 · s −1 and 3.7 · 10 −3 ± 0.6 · 10 −1 for the association and dissociation of the ouabian and the receptor, respectively. This yielded a dissociation constant of 2.5 · 10 −6 M. Scatchard plots indicate heterogeneity of ouabain binding. These have been analysed on the basis that binding occurrd at two classes of independent sites. High-affinity sites were characterized by a dissociation constant of 0.2 ± 0.1 μM and low capacity ( B max = 11.0 ± 1.2 pmol/mg protein). Low-affinity sites were characterized by a dissociation constant of 4.2 ± 1.3 μM and B max equal to 25.9 ± 2.5 pmol/mg protein. K d for the low-affinity site was not significantly different from the I 50 value of 1.12 μM, suggesting that this class of site may be associated with inhibition of (Na + + K +)-ATPase activity. Comparison of (Na + + K +)-ATPase activity and amount of ouabain bound indicate a turnover of 2645 ATP hydrolysed/site per min. It is estimated that there are in excess of 3.4 · 10 6 high-affinity sites and 8.1 · 10 6 low-affinity sites per cell (i.e., a total of 1.15 · 10 7 sites/cell). This total site density value, taken in conjunction with the turnover number, predicts rates of metabolic demand and cation translocation which are consistent with observed values.