Abstract

Rabbit myometria were homogenized and fractionated into plasma membrane (F₁), endoplasmic reticulum (F₂) mitochondria (F₃) and nuclear (F₄) fractions. Activities of 5'-nucleotidase (EC 3.1.3.5), K⁺-stimulated, ouabain-inhibited p-nitrophenyl phosphatase and cytochrome c oxidase (EC 1.9.3.1) and electron microscopy were used for characterization of different fractions. After uptake by intact tissues, ³H-ouabain and ³H-digitoxin (5 x 10^-8 M) were found to be bound mostly to F₁ of rabbit myometrium. In K⁺-free media, binding to F1 was maximal and amounted to 2.7 pmol/mg of protein. Binding of ³H-ouabain to F₁ was Na⁺-dependent and was reduced maximally by 9.2 mM K⁺ to 0.2 pmol/mg of protein, and by nonradioactive digitoxin (10^-5 M) to 0.1 pmol/mg of protein. Nonradioactive ouabain, 10^-7 M, reduced this binding to 1.1 pmol/mg of protein but the same concentration of digitoxin had no significant effect. Binding of ³H-digitoxin was also Na⁺-dependent but the total amount bound to F₁ (0.68 pmol/mg of protein) was less than the amount of ³H-ouabain bound. Also 23 mM K⁺ was required to reduce this binding maximally to 0.22 pmol/mg of protein. Nonradioactive ouabain or digitoxin (10^-5 M) reduced this binding to 0.1 pmol. The ³H-ouabain bound to isolated plasma membrane fractions was not readily washed off, but the ³H-digitoxin was removed by washing. Ouabain binding was Mg⁺⁺-, adenosine triphosphate (ATP)- and Na⁺-dependent and was reduced by K⁺. Digitoxin binding could not be shown to be Na⁺- or ATP-dependent, and K⁺ had a statistically insignificant effect. The binding of digitoxin (5 x 10^-8 and 2.5 x 10^-7 M) was Mg⁺⁺-dependent and was reduced by non-radioactive digitoxin (10^-5 M). These results suggest that ³H-ouabain preferentially binds to the E₂ form of Na⁺-K⁺-adenosine triphosphate (ATPase), and that digitoxin may bind to this form only at higher concentrations. In the case of the rat myometrium, no binding of cardiac glycosides related to Na⁺-K⁺-ATPase to either intact tissues or to the isolated plasma membrane could be demonstrated. The binding was neither Na⁺-nor ATP-dependent. K⁺ or unlabeled glycoside had no effect on this binding. Ouabain binding to rat myometrium was more labile than to rabbit myometrium.