果蠅亞硫酸基轉移酵素之選殖及特性研究

Abstract

細胞質亞硫酸基轉移酵素在生理上主要的功能在於將生物體內小分子亞硫酸化，進而調控許多生理反應與現象。許多存在於大腦的神經傳導物質或神經固醇皆為其催化受質，但對於亞硫酸基轉移酵素如何藉由調控這些神經固醇而影響大腦學習記憶的機制目前仍有相當大的爭議。因此本研究最終目的在於利用果蠅作為模式動物來探討細胞質亞硫酸基轉移酵素如何影響大腦學習記憶，但由於至今關於果蠅細胞質亞硫酸基轉移酵素的研究仍相當有限，所以本文分別從果蠅細胞質亞硫酸基酸轉移酵素本身生化特性的探討與突變種果蠅動物行為分析作為整個研究的開端。雖然以目前現有的行為研究方法並未發現所選定的亞硫酸基轉移酵素基因對於果蠅學習的影響，但從生化特性的研究上我們成功的利用大腸桿菌大量表現兩種型態之果蠅亞硫酸基轉移酵素，並且從環境酸鹼值、溫度、酵素動力學分析來比較尾端19個胺基酸差異對於該酵素之影響。另外，果蠅的細胞質亞硫酸基轉移酵素也與一些已知的酵素一樣具有基因多樣性的現象，而由這些核甘酸變異所造成的胺基酸變異甚至可能影響到酵素本身的活性與特性。最後利用蛋白質結構模擬方法，針對產生變異之各胺基酸，找出了三個有可能降低酵素催化能力之重要位置。

Sulfotransferases (SULTs) regulate many biological functions through sulfonation on biomolecules, such as neurotransmitters and neurosteroids. However, the mechanism of how sulfonation regulates learning and memory is still controversial. In this study, the main purpose is using Drosophila melanogaster as model system to investigate the function of SULTs in nervous system. There is few study and information about SULTs of Drosophila to date. Therefore, our initial approaches began from two directions. Although the enhanced learning performance was not observed in CG5431 mutant by available training program, we cloned and characterized cDNAs that encode two D. melanogaster SULTs isoforms, CG5431a and CG5431b, as well as purified isoforms that over-expressed in E.coli. The effects of carboxyl terminal peptide on optimal pH, temperature and the enzyme kinetics were investigated. In addition, the genetic polymorphisms were observed in the comparison between cDNAs of CG5431 from different fly laboratory, which might affect the activity and characteristics of the enzyme. Finally, homology modeling was used to locate the polymorphic residues that may be critical for substrate binding and catalysis.