Effect of Washout of Inducing Steroid on Γ-glutamyl Transferase Activity in the Cultured Chick Embryo Retina
نویسنده
چکیده
Glutamine synthetase (GS), as measured by its 7-glutamyl transferase (GT) activity ~, can be precociously induced in chick embryo retinas in tissue culture at least 7 days before its normal rapid elevation in o v o (Reif-Lehrer, 1968). The enzyme can be induced by a number of glucocorticoids (Chader and Reif-Lehrer, 1972), cortisol being the one most commonly employed. In such experiments , the question arises whether the continual presence of inducer is necessary to maintain the elevated enzyme level initially achieved. In some systems such as in liver-derived cells, simple washing to remove inducer leads to a fairly rapid drop in induced tyrosine transaminase activity endogenous cortisol (Reif-Lehrer and Chader, 1969). Retinas were incubated overnight (except as otherwise indicated) in medium containing 0.01 #g/ml (2.6 × 10-8 M) cortisol or [SH]cortisol (2.2 vCi/ml, i.e., 11/~Ci/flask containing one retina.) Several control samples received no cortisol. At the end of this preincubation, the retinas were washed three or four times with 5 ml of prewarmed medium, which was removed as completely as possible by suction each time. The second and/or third wash included either a 20-or 60-min incubation on the gyrotory shaker. Some retinas were harvested before and some after the washing, while the rest were put into fresh medium without cortisol (after washing) for varying periods of time up to 72 h. In some experiments, additional cultures were included in which the final medium contained cycloheximide (2 #g/ml), ac-tinomycin-D (2 ug/ml), c0rtisol (0,01 #g/ml), or combinations of these. • T..h~ ¢~tenlt to~which:~fhe .washing procedure eliminated "ste~i~J~f~i6mi:the.::sy,,~: ~as de~ermified fr0m;du!tures " removal of inducer steroid results in a 50%drop in •. .. .. contain!rig radlo~¢twe eOrtisot. Fo~ t~hese ~iaifures..r~d~0= G T a etiyity in 24,~(,K:ul-ka a.nd.-Co.hen~ 1973..). This activity of all .m.e0ia (triplicateg, 1 • "/21 ~eaclj) #nd.ali does n0tappear? tO be tile case"forGS-in the.claick: ~asfie~.(500)~i eaeh)"w)as:)de~ei-h~ified::a'fie~" filt~tion~;~o retina. Jn this system, retinas treated with steroid eliminate any-cellular debr~g. Aliquots: (500-ul) of the for 60 min or less develop very little additional GT activity in 24 h after washout of steroid. In contrast, retinas treated for 5 h or more, and then washed, develop enzyme levels approaching those of retinas left in contact with steroid during the whole of the 24-h period. The enzyme activity attained by retinas after 24 h in cortisol remains unchanged or, more often, increases by 72 h after the hormone has been washed out of the …
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ورودعنوان ژورنال:
- The Journal of Cell Biology
دوره 63 شماره
صفحات -
تاریخ انتشار 1974