小白鼠脑及肝细胞中RNA连接酶的研究

本文利用小白鼠脑或肝全细胞抽提液为酶源,Oligo A为底物,对真核哺乳动物细胞中的RNA连接酶进行研究.经测定,其抗碱性磷酸单醋酶的活性为16—49mU标准/ml.另外,还利用小白鼠脑或肝细胞核抽提液为酶源,人工合成的UpCpU和~(32)pNp为底物,用同系层析和放射自显影技术进行检测,得到清晰的连接产物自显影点.使用 DEAE-Sephadex A_(25)柱将连接产物进行了分离,并用同系层析法对连接产物做了进一步鉴定.用KOH或碱性磷酸单酯酶水解法,证明了连接方式为:3′-P→5′-OH.实验初步发现:在小白鼠脑或肝细胞核中有一与 T_4RNA连接酶不同的新酶。     

STUDIES ON RNA LIGASE ACTIVITY IN THE BRAIN AND LIVER CELLS OF MOUSE

RNA ligase in eukaryotic mammalian cells was studied by using mouse brain and livercell extracts as enzyme sources and Oligo A as substrates. RNA ligase activity was deter-mined by measuring the formation of alkaline phosphate-resistant product from 5'-~32P-termi-hated Oligoribonucleotides. Under appropriate conditions, the activity of this enzyme inbrain and liver cells may vary between 16-49 mU/ml. The joining way between donorand acceptor is 5'-P→3'-OH. Further studies wore carried out by using synthetic UpCpUand ~32pNp as substrates and crude enzyme preparations from extracts of cell unclei of brainand liver as enzyme sources. RNA ligase activity was examined by homochromatographyand autoradiography. A clear joining product was demonstrated and then isolated from thereaction mixture by DEAE-Sephadex A25 column chromatography. The eluted fractionswere identified by DEAE-cellulose thin layer chromatography. The joining product washydrolyzed either with KOH or with alkaline phosphatase, the autoradiog