Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. Partially purification of telomerase was obtained by chromatography of HeLa cell extract on DEAE-Sepharose FF,Spermine-Sepharose CL-6B and Sephacryl S-300. To dissect the human telomerase enzyme mechanism, the optimal catalytic conditions of telomerase were measured and the human telomerase activity was reconstituted using a purified hTR RNA. The optimum pH is from 6^5 to 8^5, the optimum temperature is from 35℃ to 40℃, the optimum Mg 2+ concentration is from 1 mmol/L to 2^5 mmol/L,the apparent binding constant (K m app) of telomerase for the primer is about 8^6 nmol/L. Partially purified human telomerase extract was treated with MNase to remove endogenous telomerase RNA(hTR),the telomerase activity was inactivation. Telomerase activity was restored by incubating MNase-treated extract with purified hTR. These results might facilitate the development of the telomerase enzyme mechanism.
