牛肝中氧化鯊烯-羊毛硬脂醇環化酵素的純化和其生化特性上的探討

Abstract

在固醇和三帖類天然物的生合成途徑中，2,3-氧化鯊烯環化酵素(EC 5.4.99-)所進行的環化反應一直是自然界所發現最複雜且吸引人的化學反應之一。2,3-氧化鯊烯環化酵素催化 (3S)-2,3-氧化鯊烯進行複雜的環化/重組反應。在哺乳動物和真菌中，氧化鯊烯環化酵素能將 (3S)-2,3-氧化鯊烯轉變成羊毛硬脂醇，而在較高等的植物和海藻中，氧化鯊烯則轉變成環阿屯醇。因此，我們對於這類酵素能利用單一個酵素催化步驟，即達成四至五個環狀結構以及十個鍵的同步斷裂跟生成的反應機制之研究十分感興趣。 為了對此環化機制有更進一步的瞭解，我們試著從哺乳動物牛的肝臟中純化獲得氧化鯊烯環化酵素。此環化酵素經由超高速離心、陰離子交換層析、Hydroxyapatite管柱層析以及HiTrap Heparin管柱層析，可被純化至均質(Homogeneity)。且純化得到的環化酵素在SDS-PAGE上以commassie blue或銀染色，顯示一分子量約為70 kDa的單一蛋白質帶。 此外，為了幫助牛肝中2,3-氧化鯊烯環化酵素cDNA的建立，我們希望透過生化方法確定此蛋白質N端的氨基酸序列。透過電轉漬方式將膠片上的蛋白質轉漬到PVDF膜上，進行蛋白質的N-端氨基酸定序，我們獲得連續5個氨基酸依序為精胺酸(Arg)-天門冬胺酸(Asp)-甘胺酸(Gly)-組織胺酸(His)-離胺酸(Lys)-。

2,3-oxidosqualene cyclase enzymes (OSC: EC 5.4.99-) catalyze one of the most remarkable steps in the biosynthesis of steroids and triterpenoids. The enzyme has evoked considerable interest because of the complex mechanism of the cyclization reaction it catalyzes. In mammal and fungi, (3S)-2,3-oxidosqualene cyclase undergoes an oxidosqualene-lanosterol synthase (OSC) catalyzed reaction to lanosterol. In photosynthetic plants and algae, oxidosqualene-cycloartenol synthase (CAS) transfer the same substrate to cycloartenol. In the conversion of (3S)-2,3-oxidosqualene to lanosterol, ten formal covalent changes are involved to form tetracyclic and pentacyclic products in a single biosynthetic step. To facilitate the studies on the central problem of the cyclization mechanism, we pursue the purification of OSC from bovine liver. OSC was purified to homogeneity by ultracentrifugation, anion-exchange chromatography, Hydroxyapatite and HiTrap Heparin chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of ~70 kDa, and the protein can be visualized with a commassie blue or silver stain. In addition to the ongoing molecular biology approaches to determine the cDNA of 2,3-oxidosqualene cyclase of bovine liver, we also pursue the biochemical approach to characterize the N-terminal amino acid sequence. By electroblotting method, we transferred the protein to PVDF membrane from SDS-PAGE and obtained the 5 amino acid sequence Arg-Asp-Gly-His-Lys- in order.