甘藷塊根澱粉磷解酶L78之性質分析

Abstract

本實驗室研究發現，甘藷塊根的澱粉磷解脢 (L-SP) 分子構造比肝糖磷解脢多出一段近百個胺基酸的片段 (L78)，因而發現具有不需醣引子合成直鏈醣之活性 (primer independent activity, PI)。此催化機制可能經由L78P上的Lys與Glc-1-P結合，L78P扮演醣引子的角色以啟動澱粉合成反應；因為若L-SP失去L78P (L-SP*) 就不再有此種PI活性。此外，L78P降解與否，可能調控L-SP的作用方向。本論文的主題乃針對L-SP的PI催化機制，進一步確認L78P是否會透過Lys與Glc-1-P結合，以及L78P是否能救回上述L-SP*的PI活性。 本研究發現，L78P表現蛋白質在原態電泳出現數個色帶，推測應為表現所得的L78P構造不穩定，因而具有不同構形所造成；由於L78P分子並不大，可能缺乏完整的二級結構，因而無法在CD光譜得到明確的結果，而呈現random coil。加入磷酸酶CIAP去磷酸的實驗證實，L-SP以及L78P可降低磷酸釋放量；以Lys加入競爭發現，Lys能與L-SP競爭Glc-1-P，抑制L-SP合成直鏈醣而釋放磷酸的能力，顯示L-SP是利用L78P上的Lys與Glc-1-P結合。外加L78P到L-SP*中發現，其合成直鏈醣生成物 (磷酸以及澱粉) 含量都有上升，顯示L78P具有救回L-SP* PI活性的能力。另外，活化態的proteasome (proteasome deletion mutant) 確實可降解L-SP以及L78P，可能藉由泛素系統來調控L-SP的催化活性。

When compared with glycogen phosphorylase, it was found that starch phosphorylase (L-SP) from sweet potato roots has an insertion containing 78 amino acids (L78P) which might involve in the primer-independent (PI) activity of L-SP, since L-SP lost this PI activity totally if the L78P was removed by proteolysis (L-SP*). It was postulated that L-SP might bind with Glc-1-P at Lys residues on its L78P that might play the role of primer in starch synthesis. It was also demonstrated that L78P could control the direction of L-SP catalytic reactions by prtoein degradation. This study explored the structural features of L78P, and examined the possibility of the binding between L-SP and Glc-1-P. Besides, L78P was added to L-SP* for the rescue of its PI activity We found that L78P have four bands on native-PAGE which might be caused by its flexible conformation. From the circular dichroism (CD) spectroscopy analysis, L-SP was identified as random coil. When we added calf intestinal alkaline phosphatase to release the phosphate of Glc-1-P, L-SP and L78P could inhibit the reaction and decrease the phosphate leval. In addtion, Lys could compete with Glc-1-P in binding to L-SP; and the ability of L-SP to synthesize starch would be blocked by Lys. These results showed that L-SP can bind to Glc-1-P at the Lys residues of L78P. We also found that after adding L78P to L-SP*, the phosphate releasing leval increased and the starch synthesis began. In addition, it was found that proteasome (proteasome deletion mutant) could degrade L-SP and L78P; this observation indicated that L-SP activity might be regulated via ubiqutin-proteasome system.