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.
Footnotes
- Received February 5, 1973.
- Accepted November 15, 1973.
- © 1974 by The Williams & Wilkins Co.
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