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36 



24 



12 



uptake of hormones into 

 calf endometrium cells 



h'-estradiol 



h'-progesterone 



h'-testosterone 



h'- HYDROCORTISONE 



Fig. 15. 



40 



30- 



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UPTAKE OF HORMONE INTO 

 CRUDE CHROMATIN FRACTION 



H^-ESTRADIOL 



H -progesterone 



h^-testosterone 

 1 , h'-hydrocortisone 



Fig. 16. 



lower so it makes the interpretation of that re- 

 sult rather more difficult. 



In the final figure of this trio we see the 

 specific activity of hormone actually bound (fig. 

 17). I define "bound" as that hormone which 

 can be centrifuged through a sucrose gradient 

 along with the chromatin into the pellet and 

 which isn't removed by subsequent exhaustive 

 dialysis. Again, specific activity of the incorpo- 

 ration of the target tissue specific hormones is 

 high and that of testosterone and hydrocortisone 

 relatively low. More recent experiments dem- 



onstrated an even more dramatic effect with an 

 equivalent technique. 



GRUN: What tissue was this in? 



CHALKLEY: This is the calf endometrium, 

 the epithelial layer of the uterus, which had 

 been scraped off and incubated. 



SCHRAER: Do you analyze it for the hor- 

 mone or just for the label? 



CHALKLEY: Just for the label.* 



SCHRAER: Do you know if it has hormones 

 in those parts of the cells? 



CHALKLEY: We put in labeled hormone and 

 later on we follow the behavior of the counts. 

 Now, it may be that this is being degraded and 

 then the degraded material is being bound. So 

 far we haven't found that out. 



In Table V we see the effects of very dif- 

 ferent treatment on the chromat in containing 

 the hormone. Organic solvents appear to rea- 

 sonably efficiently extract a hormone. Sulfuric 

 acid (2N) which we know will extract histones 

 virtually quantitatively, also solubilized a por- 

 tion of the counts. Guanidinium chloride, which 

 we also know dissociates histones and de- 

 natures proteins, released over 50%. Sodium 

 chloride (2 M) released only a small fraction of 

 these counts. However, the real clue to the pos- 

 sibility of binding to histones was given, first 

 of all, from histones isolated from this and put 

 through the IRC -50 column. Not a single count 

 above background was found and in fact essen- 

 tially all of the hormone, after treatment with 

 acid, was fully dialyzable. I should add, also that 

 recently we've found that the hormone appears 

 to be thermolabile. In 30 minutes at 37° about 

 50 to 60% of the hormone can be thermolabil- 

 ized (13). 



As a further check on what the binding 

 really involved we suspended the chromatin in 

 2.09 M cesium chloride, which is of sufficiently 

 high ionic strength to dissociate the histones and 

 other chromosomal proteins, and then centri- 

 fuged the solution at high speed. You can actually 

 band the histone component (14) (Fig. 18). The 

 bulk of the non-histone protein bands at a lower 

 density than the histone. This material aggre- 

 gated as a very, very thin skin in the tube, A 

 considerable number of the counts were localized 

 in this skin which we homogenized and counted. 



EPEL: Before you go into this new subject, 

 could you clarify the conclusion from the 

 estradiol experiment? 



•Subsequent studies have demonstrated that 99% of the 

 bound h3 was present as unchanged estradiol- 17>9 (13). 



142 



