Abstract
核糖核酸還原脢是一種轉換核糖核酸雙磷酸成去氧核糖核酸的酵素。該酵 素是由兩個次單位 M1 及 M2 所組成,其中 M2 上含有一個酪胺酸自由基 及一個含鐵單元。一種 DNA合成抑制劑－氫氧尿素， 可以破壞 M2 酪胺 酸上的自由基，而該自由基對核糖核酸還原脢的活性具有重要的影響， 這種破壞同時造成鄰近的含鐵單元不穩定而釋出鐵離子。 目前為止，對 氫氧尿素具有抗性的細胞株已經從老鼠 L 細胞被選殖出來， 此類細胞株 具有過量的核糖核酸還原脢,並且貯鐵蛋白的含量也增高了。 在中國倉鼠 卵巢細胞株 (CHO) K1 中，貯鐵蛋白 mRNA 的表現較其 X- 射線敏感的突 變株xrs-5來的高。 為了了解核糖核酸還原脢的過量形成以及鐵蛋白表現 之間的關係， 我們由 CHO K1 及 xrs-5 篩選出來了抗氫氧尿素細胞株 K1/H 和 xrs-5/H。我們證實這兩抗藥細胞株對氫氧尿素均 比其親代細胞 更具有抗性， 同時也有更高的核糖核酸還原脢的活性及更多的 mRNA 表 現。 在貯鐵蛋白的表現上， 藉由北方及西方墨點法發現這兩抗藥細胞株 中的貯鐵蛋白 mRNA含量與其親代細胞的表現是相同的,而在貯鐵蛋白蛋白 質表現上, K1/H 中的含量較 K1 多了兩倍,但在 xrs-5 及 xrs-5/H中的 含量則是相同的而且表現都較 K1 來得低。 為了證實突變株中貯鐵蛋白 的表現是如正常一樣, 仍然受到鐵離子的調控， 我們利用檸檬胺鐵來處 理所培養的細胞，發現xrs-5貯鐵蛋白的表現的確是與 K1 一樣受到鐵離 子的影響。 本篇研究說明了核糖核酸還原脢的過量表現和貯鐵蛋白的表 現並無明顯的關係,可能有著另一些調控鐵離子途徑的存在，用以調整 CHO 細胞株中核糖核酸還原脢所釋出的過量鐵離子。 Ribonucleotide reductase (RR) is an enzyme which converts ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The enzyme consists of two subunits M1 and M2. Hydroxyurea, an inhibitor of RR, destroys the M2 tyrosyl free radical which is essential for RR activity and destabilitizes M2 iron center. Hydroxyurea resistant cell lines from the mouse L cell line have been isolated and were shown to have elevated level of RR and ferritin. The level of ferritin mRNA in CHO K1 cells was 8 fold higher than in its x-ray sensitive mutant xrs-5, but the RR activity was the same in those two cell lines. Hydroxyurea resistant cell lines K1/H and xrs-5/H were selected from CHO K1 and xrs-5 cells. These two resistant cell lines had similar RR activity and M2 mRNA level, and both are higher than their parental CHO K1 and xrs-5 cells. The mRNA for ferritin H chain in the hydroxyurea -resistant cells was the same as their respective parental cells. The ferritin protein in K1/H cells was 2 fold higher than in CHO K1, but was the same in the xrs-5/H and xrs-5 cells. Treatment with ferric ammonia citrate increased the level of ferritin protein in both CHO K1 and xrs-5 cells, indicating that the ferritin mRNA in both cell lines retains the normal property of being regulatable by iron. The results suggest that overproduction of RR is not accompanied by ferritin expression, and there is another pathway of regulation of excess free irons released from the M2 subnuit of RR in CHO cells.