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標題: | 哺乳動物核醣核酸水解?抑制劑之純化與性質研究 Purification and Properties of Mammalian RNase inhibitors |
作者: | CHEN.SHU-JEN 陳淑珍 |
出版年 : | 1986 |
學位: | 碩士 |
摘要: | 經過硫胺?析及親和性層析二步驟,從豬肝分離純化出核醣核酸水解?抑制劑。由500克新鮮豬肝中約可得到1.5毫克的抑制劑,純化倍數將近60,000倍,回收率約為總活性的13%,專一活性(specific activity)是每毫克蛋白質90,000單位。SDS-PAGE分析,有一主要呈色帶(major band),分子量估計為50,000,含有高量白胺酸(leucine),每分子半光胺酸或光胺酸(cysteine or cystine)的總和約為33個,且硫胺基(sulfhydryl group)是維持抑制劑活性所必須。 又以同法純化豬腦及牛肝中的抑制劑。由SDS-PAGE結果,得知其分子量略大於豬肝中的抑制劑,約53Kd.以 try-psin將其各別水解,經由HPLC作勝?圖譜分析,進一步說明三者的相異性,所以同種動物不同組織或不同動物同一組織所含抑制劑並不完全相同。 我們將豬生長激素由腹腔打入剛離乳的小白鼠在不同時間後分析肝臟萃取液中核醣核酸水解?及抑制劑的活性。在打入生長激素24小時後,抑制劑活性有明顯上升趨勢,而核酸水解?總量卻呈下降趨勢,因為生長激素的標的器官是在肝臟,作用是引發肝臟蛋白質合成。由初步研究結果,我們推測核酸水解?和抑制劑的相互作用可能和肝臟甚至其他器官中蛋白質合成的調節有關。 The RNase inhibitor (RI) was purified from porcine liver by ammonium sulfate fractionation (35-60%) followed by affinity chromatography on an RNase A-Sepherose column. This two-step operation could yield 1.5mg of purified RI from 500g of fresh pork liver with 60,000-fold purification and a yield of 13% of total RI activity. The specific activity of this purified RI revealed 90,000 unit/mg protein one unit being the amount of RI needed to achieve 50% inhibition of 5ng of bovine pancreatic RNase A. Biochemical analyses showed the following properties: the molecular weight is around 50 KDa (SDS-PAGE); it contains high percentage of leucine and cysteine and cyatine (about. 33 residues determined as cysteic acid per molecule); sulfhydryl groups are essential for the RI activity. RI was also purified from porcine brain, spleen and bovine liver. SDS-PAGE patterns of these RI’s showed slightly larger size, about 53KDa, Tryptic peptide mapping by using C18 reverse phase HPLC also indicated structural differences among these RI’sfrom different tissues and species, implying the existence of tissue and species heterogeneity. Porcine growth hormone was administered intraperitoneally into 4-week old mice and the liver extract was analyzed for RNase and RI activities. It was found that RNase activity drcreased until 24 hours after growth hormone treatment. On the other hand, RI activity was elevated. It is well known that growth hormone exerts its effects through binding to the receptor on liver membranes and then increasing protein synthesis in the liver. From our preliminary result, it is very likely that the RNase-RI system plays an important role in the regulation of protein biosynthesis in liver and probably other tissues as well. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75591 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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