甘藷塊根L型澱粉磷解脢激脢之純化與性質分析

The Purification and Functional Assay of L-Form Starch Phosphorylase Kinase from Sweet Potato Roots

楊光華(Guang-Huar Young)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.01125

摘要

甘藷塊根L型澱粉磷解脢 (L-SP, EC 2.4.1.1) 中央序列 (L78) 斷裂後，可提高對於澱粉的親和力。以電腦程式PC/GENE分析L78，預測此序列包含有一段易受到蛋白脢攻擊的PEST site以及數個可能受到磷酸化修飾的位置。本論文利用純化自甘藷塊根L-SP作為基質，篩選甘藷塊根蛋白質中可對L-SP進行磷酸化作用的激脢。根據專一性免疫沈澱法的實驗結果顯示：L-SP確實可受到甘藷塊根中內生性鎂或錳需求型蛋白質激脢的磷酸化修飾，因此利用硫酸銨分劃以及三種不同性質的管柱層析法，將此一激脢自甘藷塊根粗抽液中純化分離至部份均質，命名為L型澱粉磷解脢激脢 (LSK)。將LSK以Native/SDS二次元電泳解析，再配合質譜儀鑑定，發現當中含有顯著Ser/Thr蛋白質激脢訊號。此一激脢的原態分子量，經膠體過濾法以及膠體內激脢分析法估計為338 kD。LSK的活性可受到蛋白質激脢抑制劑staurosporine的抑制，而L-SP的磷酸化可被來自於牛腸胃道的鹼性磷酸脢去磷酸化。使用不同的L-SP片段作為LSK的基質，發現LSK針對含有L-SP中央序列的表現蛋白質L78P作用。磷酸化胺基酸分析結果顯示LSK是針對L78序列的Ser進行磷酸化修飾。針對L78P正常株的定點突變結果，則確認LSK針對L78的Ser527進行磷酸化修飾。磷酸化的L-SP在試管中的結果顯示：L-SP的磷酸化修飾會導致L78的移除，但是並不會改變本身的酵素動力學參數。根據本論文結果顯示：LSK對於L-SP的磷酸化修飾在澱粉代謝中扮演著重要的調節性角色。

關鍵字

甘藷 ； 蛋白質激脢 ； 蛋白質磷酸化 ； 甘藷塊根 ； 澱粉磷解脢

並列摘要

The degradation of middle chain (L78) of L-form starch phosphorylase (L-SP, EC 2.4.1.1) from sweet potato roots could increase the binding affinity to soluble starch. The computational analysis of the amino acid sequence on L78 predicted that it contains a proteolytic PEST site and several phosphorylation sites at its Ser residues. We used purified L-SP from sweet potato roots as a substrate to screen the relative protein kinase(s). According to the results of specific immunoprecipitation, we demonstrated that L-SP was phosphorylated in Mg2+ or Mn2+-dependent manner by endogenous protein kinases from sweet potato roots. The protein named L-form starch phosphorylase kinase (LSK) was partial purified from sweet potato roots by ammonium sulfate fractionation, followed by ion-exchange, phosphocellulose, and gel-filtration chromatography. LSK was resolved in a two dimensional native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system and characterized using liquid chromatography-tandem mass spectrometry. The native molecular mass of the enzyme determined by gel filtration and in-gel kinase assay was 338 kD. The activation of LSK was suppressed by the protein kinase inhibitor staurosporine, and the phosphorylation of L-SP was abolished by calf intestinal alkaline phosphatase. An expressed peptide (L78P) containing the essential part of L78 was phosphorylated by the kinase, but the proteolytic modified L-SP which lost its L78 fragment was not. Phospho-amino acid analysis showed that only serine residues were phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at serine residues on L78, we demonstrate that only Ser527 on L78 is phosphorylated by the kinase. In vitro assays of phosphorylated L-SP which was mixed with crude extract from sweet potato roots show that the proteolytic degradation of L78 was introduced by L-SP phosphorylation, but had no change in its kinetic parameters. These results suggest that the phosphorylation of L-SP by LSK plays an important regulatory role in starch metabolism.

並列關鍵字

Ipomoea batatas ； Protein kinase ； Protein phosphorylation ； Sweet potato roots ； Starch phosphorylase

參考文獻

張世宗 (1999) 甘藷塊根chaperonin及proteasome之分離與性質研究 博士論文 國立台灣大學 台北

Ahmad Z, Huang KP (1981) Dephosphorylation of rabbit skeletal muscle glycogen synthase (phosphorylated by cyclic AMP-independent synthase kinase 1) by phosphatases. J Biol Chem 256: 757-760

Akazawa T, Nelson OE (1966) Starch granule-bound adenosine diphosphate glucose-starch glucosyltransferases of maize seeds. J Biol Chem 241: 2280-2285

Albrecht T, Koch A, Lode A, Greve B, Schneider-Mergener J, Steup M (2001) Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization. Planta 213: 602-613

Albrecht T, Greve B, Pusch K, Kossmann J, Buchner P, Wobus U, Steup M (1998) Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies. Eur J Biochem 251: 343-352