One of the general transcription factors, TFIIA, was purified to homogeneity from HeLa cell nuclear extracts by yeast TFIID affinity chromatography. Human TFIIA had a molecular weight of approximately 38 kd. It was able to associate with the complex formed by yeast TFIID and the TATA elements of the adenovirus E4 and ML promoters, and the HSP70 promoter. The association extended the protected region on each TATA element by yeast TFIID from DNase I digestion. Affinity‐purified TFIIA was also able to stimulate transcription from the E4 and ML promoters in in vitro reconstituted systems.
The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E4) promoter by binding to specific promoter elements. Among the multiple forms of ATF/E4TF3, two forms with molecular masses of 47 and 43 kDa, which are most active in transcription in vitro from the E4 promoter, have been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and biochemically characterized. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence, but not in the presence, of the specific DNA sequence. All of these dimers were able to activate transcription in vitro from the E4 promoter by binding to the specific DNA sequence. Their activities to bind to DNA or stimulate transcription were different. The ability of the 47-kDa homodimer to stimulate transcription in vitro from the E4 promoter was approximately nine and three times higher than the abilities of the 43 kDa homodimer and the heterodimer, respectively, at the same level of DNA-binding activity. However, the affinity of the 47-kDa homodimer for DNA was lower than that of the 43-kDa homodimer, and the heterodimer had intermediate affinity. These results are the first to show differential binding and transcriptional activation activities of the different dimers of ATF/E4TF3, using purffied cellular proteins rather than cloned gene products.
Soluble Ca 2+ -activated ATPase was extracted from the calcareous alga Cricosphaera roscoffensis var. haptonemofera, and purified approximately 20-fold by DEAE-cellulose chromatography. The ATPase was characterized äs follows.The enzyme is activated by several divalent cations such äs Ca 2+ , Mg 2+ , Mn 2+ and Co 2+ , the most effective being Ca 2+ . Sr 2+ , Ba 2+ and La 3+ do not activate the enzyme. In the presence of 3 mM ATP, maximal enzyme activity is obtained at approximately 9 mM Ca 2+ . At the optimal Ca 2+ concentration, addition of Mg 2+ or La 3+ gives a strong inhibitory effect. Enzyme activation by Ca 2+ is not affected by Na + or K + .The optimal pH for Ca 2+ activation of the enzyme is approximately 9.6.The enzyme hydrolyzes several nucleoside tri-and diphosphates, but ATP serves äs the best Substrate with an apparent Km of 0.7 mM. /3-Glycerophosphate and p-nitrophenylphosphate are hardly hydrolyzed. The enzyme is inhibited by EDTA, ethacrynic acid, 7V-ethylmaleimide andp-CMB, but not by ouabain, Oligomycin, 2,4-dinitrophenol and DCCD. The enzyme is very unstable at 4 °C, but glycerol partially prevents inactivation of the enzyme.Noticeable activity of Ca 2+ -activated ATPase is also observed in the microsomal fraction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.