Interactions of gonadotropins with corpus luteum membranes VIII. The different properties of rat luteal cell light and heavy membranes cannot be explained by fractionation of inside-out and outside-out plasma-membrane vesicles

Abstract

Fractions enriched in luteal cell plasma-membranes were isolated from superovulated rat ovaries. Cell surface-membrane marker enzyme activities were measured in both light and heavy membrane fractions, with or without treatment with various detergents, to assess whether these fractions consisted of resealed vesicles with different orientations (i.e. inside-out or outside-out). Control and digitonin-treated membranes were also fractionated on sucrose-density gradients to monitor the buoyant density perturbation of each surface-membrane marker induced by this detergent. In another experiment, hCG-binding sites of superovulated rat ovaries were labelled in vivo by injecting [ 131I]hCG intravenously 2 h prior to fractionation and isolation of ovarian membranes. Membranes were then tested for the ability to bind [ 125I]hCG and [ 131I]hCG in vitro. These experiments indicated that light and heavy rat luteal cell membrane fractions had similar levels of inside-out membrane vesicles (ranging from 0 to 34%) and that the hCG-binding sites of the 2 fractions appeared to be equally freely accessible to hormone in vivo. It is concluded that differences in membrane orientation cannot account for the distinct properties and marker enzyme profiles of rat ovarian light and heavy membranes, providing further support that these membrane are derived from different regions of the luteal cell surface-membrane.