Abstract
RC-RNase 是一種自牛蛙蛙卵卵黃顆粒中純化出來的嘧啶-鳥嘌呤序列特異性的(pyrimidine-guanine sequence-specific)核醣核酸水解酵素(Ribonuclease;RNase)。具有抗癌特性,且會誘發 HeLa cell之細胞自戕(Apoptosis)。除了RC-RNase,牛蛙蛙卵尚有暫稱為Up、Low、Ha、Hb、Hc五種主要的核醣核酸水解酵素。在本篇論文中,我們利用phosphocellulosecolumn(P11),carboxylmethyl cellulose column(CM52),Blue dextranSepharose CL-6Bcolumn(Blue column)等層析管柱及FPLC MonoS層析管柱,分別純化這幾種核醣核酸水解酵素。並以anti RC及anti Up兩種抗體進行西方轉印法檢測,發現RC-RNase不易被anti Up抗體辨視出來,而Hb則皆不會被這二種抗體辨識。由RNA凝膠活性測試,我們發現各種RNase活性強弱依序為RC>Ha>Up>Hb>Hc>Low。而在純化這些RNase時發現Up、Low、Hb三種蛋白質的相對含量並不穩定,隨著不同批次蛙卵而有變異,推測可能與牛蛙蛙卵之成熟度有關。而純化所得之Ha、Hb、Hc三種蛋白質,以溴化氰切割,各得到一主要多胜鏈,分別進行胺基酸序列分析。Ha的部分胺基酸序列為PTSLFD(D)KD(C)NTFIYSLPGP/CVKALC,Hb的部分胺基酸序列為GNTIFIVDGQCKKFN,Hc之部分胺基酸序列為DNTIFIVDGQ(C)KKDFNDI-D。其中Ha之部分胺基酸序列,與我們先前所得到的一個c-DNA clone所推出的胺基酸序列,幾乎完全相同。而Ha、Hb、Hc的部分胺基酸序列與牛胰臟RNA水解酵素(RNase A)群之序列,亦有相似性。RC-Rnase is a pyrimidine-guanine sequence-specific ribonucleaseisolated from the yolk granules of bullfrog (Rana catesbeiana).The RNase possesses antitumor activity and could induce theapoptosis of HeLa cell. BEsides RC-RNase, thereare at least fivemajor RNase, celled Up、Low、Ha、Hb and Hc, exist in thebullfrog oocytes. In this thesis, we used phosphocellulosecolumn (P11), carboxylmethyl cellulose column (CM52), Bluedextran Sepharose CL-6B column (Blue column) and FPLC Mono S topurify those RNase. Antibodies anti RC and anti Up were raisedin rabbits for Western blotting analysis. RC-RNase was weaklydetected by anti Up antibody, and Hb protein was not detectableby both antibodies. The relative specivic activity of theseRNase was RC>Ha>Up>Hb>Hc>Lowdetermined by RNA-casting gel assay.The relative amount of Up, Low and Hb was not constant in eachbatch of oocytes, which mayge due to differential maturity ofbullfrog oocytes. The Ha、Hb、Hc proteins were digested withcyanogen bromide,then the major polypeptide was pruified andsequenced. The parital amino acid sequence of Ha is PTSLFD(D)KD(C)NTFIYSLPGP/CVKALC, that of Hb is GNTIFIVDGQCKKFN, and thatof Hc is DNTIFIVDGQ(C)JJDFBDU-D. The partial amino acid sequenceof Ha is identical to the translated sequence of one c-DNA cloneisolated in this laboratory. The amino acid sequence of Ha、Hb、Hc also show similarity to that of that of the RNase Asuperfamily protein.