1. A new method is described for the titration of membranes with steroid hormones, and the binding curves for male and female smooth rat liver microsomes with oestradiol and testosterone are reported.2. At least three classes of binding site exist, including one group to which the hormones are bound highly specifically and with very high affinity.3. Smooth vesicles of the male or female bind, respectively, approximately 25-30 nmoles of oestradiol or testosterone per gram of membrane protein, but only 0-5 nmoles per gram of the other hormone. Testosterone does not occupy the oestradiol sites of male membrane, or oestradiol the testosterone sites of female membrane.4. Aflatoxin B, completely destroys the high affinity binding sites for oestradiol on male membrane, and for testosterone on female membrane. This effect of the toxin can be prevented by pretreatment of male smooth microsomes with oestradiol but not testosterone, and female membranes with testosterone but not oestradiol.5. Parallelism between the physicochemical study reported here and previously published data strongly suggests that these high affinity sites are involved in polyribosome-microsomal membrane association.The binding of polysomes to smooth microsomal membranes of rat liver can be promoted in vitro by the presence of certain steroid hormones [l]. The formation of an artificial rough membrane was monitored by assaying a membrane bound disulphide interchange enzyme, the activity of which is masked by polysome binding, and the results were confirmed by electron microscopy [2]. The hormone requirements for membrane-polysome association are remarkably sex-specific : oestradiol promotes association of male rat liver smooth microsomes with male polysomes, but testosterone has no effect ; testosterone promotes the binding of female polysomes to female smooth microsomes, but oestradiol has no effect [3].Aflatoxin B, degranulates both male and female rat liver rough microsomal membranes, and destroys the ability of smooth membranes to bind polysomes in the presence of the required steroid [3]. These findings indicate that the polysome-membrane interaction may be controlled by the binding to male microsomes of the "female" steroid hormone, and vice versa.The existence of sex-specific, high affinity binding sites for oestradiol in male rat liver smooth membranes and for testosterone in female smooth membranes has been demonstrated directly by titrating the vesicles with n-[2,4,6,7-3H4]oestradiol or [4-14C]-testosterone. Smooth male microsomes possess a far smaller number of high affinity sites than female smooth membranes for testosterone, and similarly, female microsomes have far fewer high affinity sites than male membranes for oestradiol. Furthermore, the presence of one hormone does not alter the binding curves of the other. These preliminary findings [4] confirm the existence of sex-specific, high affinity binding sites indicated by the polysome-binding experiments described above [1-31. This communication reports more detailed studies of the pr...
1.Smooth reticular membranes from male rat liver contain high affinity binding sites for 2. Rough membranes from which polysomes have been removed by EDTA (degranulated 3. Ethyl acetate extracts of degranulated membranes contain materials which block the high 4. These extracts promote the at,tachment of ribosomes to smooth membranes of the same but 5. The ethyl acetate extracts from male and female degranulated membranes contain suboestradiol. Similar membranes from females contain analogous sites for testosterone. membranes), contain no high affinity binding sites for either hormone. affinity binding sites on smooth membranes of the same but not the opposite sex.not the opposite sex.stances which behave like oestradiol and testosterone, respectively.Polysomes can be removed from rough reticular membranes by treatment with EDTA, and the resultant degranulated membranes will rebind polysomes from the same, but not the opposite, sex in presence of magnesium ions [l-31. Smooth membranes, in contrast, will not bind polysomes unless a steroid hormone is also added. The steroid hormone requirements are sex-specific : oestradiol promotes binding of male polysomes to male smooth membranes and testosterone has a similar interaction in the female, in vitro [l-31. Female polysomes will bind to male smooth or degranulated rough membranes in presence of testosterone, and male polysomes to female membranes in the presence of oestradiol [2]. Oestradiol or testosterone can be replaced in the binding experiments by ethyl acetate extracts of female or male polysomes, respectively, [2]. A similar extract of male rough reticulum can be substituted for both testosterone and oestradiol, acting as a mixture of these two hormones [2]. It is possible that the basic difference between smooth and degranulated rough membranes from male rat liver is that the latter membranes already contain oestradiol (or a related substance callcd "0-substance" in [2]). Similarly female degranulated rough membranes would contain testosterone or "T-substance". Consistently, it has been shown that female smooth membranes possess "high-affinity" sites for testosTrivial Names. Cholesterol, 5-cholesten-3@-01; testosterone, 17/3-hydroxy-4-androsten-3-one.20 Eur. J. Biochem., Vol.29 terone but not oestradiol, and male smooth membranes contain analogous sites for oestradiol but not testosterone [4,5].I n this paper, we present data on the binding of steroid hormones and polyribosomes to degranulated rough microsomes from male and female rat liver. We show that materials can be extracted from degranulated rough membranes which block the "high affinity" steroid binding sites on, and cause polyribosome binding to, the smooth membranes of the same, but not the opposite, sex. MATERIALS AND METHODS Smooth and Rough Microsomal FractionsThese were prepared from the livers of 200g albino rats by differential centrifugation on a sucrosedensity gradient as described previously [5]. Degranulated Rough MembranesRough microsomal membranes were resuspended in 0.25 M sucroae s...
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