24] Techniques for monitoring the distribution of the estradiol-binding protein complex between cytoplasm and nucleus of intact cells

Abstract

Publisher Summary A number of observations have led to a hypothesis in which the initial step in the stimulation of uterine growth by estradiol (E) is the interaction of the hormone with specific binding proteins or receptors (B) in the cytoplasm of uterine cells. The estradiol-binding (EB) protein complex thus formed is subsequently transferred to the cell nucleus. The methods described in this chapter examine the formation of EB and its distribution between cytoplasmic and nuclear fractions of immature rat uteri. The chapter considers, as an example, the situation at the conclusion of an in vitro incubation of the uterus with [3H] E or an in vivo treatment with the hormone. The labeled hormone is present in several states within the tissue as (1) EB complexes in cytoplasmic and nuclear fractions (specifically bound E); (2) complexes with proteins other than the specific estradiol-binding proteins (nonspecifically bound E or EP); and (3) E free within the cells, in extracellular spaces, and partitioned into lipid components of the tissue (trapped E). Similarly, when a particular amount of EB is present, a defined amount of unfilled specific binding sites will also be present within the uterine cells. The distribution of E among these components will be a function of E concentration, incubation time, temperature, the quantity of each component present, and the relative affinity each component exhibits for E. For an examination of EB as a function of time or its distribution between cytoplasmic and nuclear fractions, several requirements must be met. (1) It must be possible to stop the formation of EB and preserve the EB formed to that point. (2) Methods must be available to separate cytoplasmic and nuclear fractions. (3) Methods must be available to assay bound E in these fractions. (4) It must be possible to distinguish EB from EP.