The effects of Mg2+ ions or EDTA on nuclear integrity and apparent subcellular distribution of unoccupied oestrogen receptors in breast cancer cells

Homogenisation and fractionation of cells in the presence of Mg2+ or EDTA resulted in unoccupied oestrogen receptor being recovered in the particulate fraction. Nuclei were partially purified by pelleting at 100,000 g through 41% and 44% (w/w) sucrose (in buffer containing Mg2+ or EDTA), plasma membranes being collected from the top of the 41% barrier. In Mg2+-prepared fractions, both 5'-nucleotidase and unoccupied receptor were distributed between plasma membrane, partially-pure nuclei and mitochondrial/microsomal pellets. Lactate dehydrogenase was not a significant contaminant of particulate fractions. In EDTA fractions, the majority of binding activity was in the partially-pure nuclei (which were extensively disrupted) and mitochondrial/microsomal pellets. Little or no binding was found in the EDTA-prepared plasma membranes which were amorphous in appearance. Mg2+-prepared nuclei, freed of membranous contamination by pelleting through 1.8 M sucrose, were intact by electron microscopy but had no 5'-nucleotidase or unoccupied receptor. These data suggest that recovery of receptor in partially-pure nuclei during fractionation is not caused by trapping of cytosolic protein but rather by redistributed nuclear receptor having become bound to adhering plasma membrane fragments during homogenisation. Implications for the study of cell-free systems are discussed.