Diversity in enzyme localization: difference in Tetrahymane and Paramecium AK enzymes.

• Project/Area Number: 15K07151
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Animal physiology/Animal behavior
• Research Institution: Kochi University
• Principal Investigator: Suzuki Tomohiko 高知大学, 教育研究部自然科学系理工学部門, 教授 (60145109)
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: アルギニンキナーゼ / 酵素の局在 / 基質阻害メカニズム / プレニル化 / ミリストイル化 / RNAi / 基質阻害の速度論 / ゾウリムシ / 繊毛運動 / Paramecium

Outline of Final Research Achievements

The cDNAs for arginine kinases (AK1, AK2, AK3 and AK4) from Paramecium tetraurelia were isolated, and their recombinant enzymes were expressed in E. coli. The AK1, AK2 and AK4 showed a typical Michaelis-Menten profile, while the AK3 displayed a substrate inhibition profile. The latter inhibition was caused by a formation of SES complex, which is formed by additional substrate to the normal ES complex. The AK3 enzyme was synthesized using a cell-free protein synthesis system, and the resultant enzyme was purified and subjected to PMF analysis. The prenylated C-terminal peptide was clearly observed. Western blot analyses indicated that the AK3 is localized in the ciliary fraction. The role of P. tetraurelia AKs on ciliary movement was examined by feeding RNA interference (RNAi) method. The swimming velocity of AK1- and AK3-silenced cells was significantly decreased to half of the control cells. In contrast, that of AK2-silenced cells was unchanged.

Academic Significance and Societal Importance of the Research Achievements

生命活動における酵素の局在化は，生理機能の維持に重要な役割を果たしている．この局在化が妨げられると酵素は本来の行き場を失う． 我々は，繊毛虫Tetrahymenaのアルギニンキナーゼ（AK）が，N末端のミリストイル基を介して繊毛膜に局在していることを明らかにした．一方で，繊毛虫Parameciumでは事情が大きく異なる．後者のAKはN末のミリストイル化シグナル配列を欠き，代わりにC末端にプレニル化シグナル配列を持っていた．つまり，両者は全く異なる方法でAK酵素を繊毛膜に局在させている可能性が高い．この研究では，繊毛虫AK酵素の局在化メカニズムの多様性を明らかにし，生理機能との関連性を探る．

Research Products

• [Journal Article] Kinetic analyses of the substrate inhibition of Paramecium arginine kinase. (2018)
  • Author(s): Yano, D. and Suzuki, T.
  • Journal Title: Protein Journal Volume: 37 Issue: 6 Pages: 581-588
  • DOI: 10.1007/s10930-018-9798-2
  • Peer Reviewed
• [Journal Article] Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism. (2017)
  • Author(s): Yano, D., Suzuki, T., Hirokawa, S., Fuke, K., and Suzuki, T.
  • Journal Title: Int. J. Biol. Macromol. Volume: 101 Pages: 653-659
  • DOI: 10.1016/j.ijbiomac.2017.03.133
  • Peer Reviewed
• [Journal Article] Arginine kinases from the precious corals Corallium rubrum and Paracorallium japonicum: presence of two distinct arginine kinase gene lineages in cnidarians. (2017)
  • Author(s): Matsuo, T., Yano, D., Uda, K., Iwasaki, N., and Suzuki, T.
  • Journal Title: Protein J. Volume: 36 Issue: 6 Pages: 502-512
  • DOI: 10.1007/s10930-017-9745-7
  • Peer Reviewed
• [Journal Article] Evidence for N-terminal myristoylation of Tetrahymena arginine kinase using peptide mass fingerprinting analysis. (2016)
  • Author(s): Motomura, S. and Suzuki, T.
  • Journal Title: Protein J. Volume: 35 Issue: 3 Pages: 212-217
  • DOI: 10.1007/s10930-016-9663-0
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Arginine kinase from Myzostoma cirriferum, a basal member of annelids. (2016)
  • Author(s): Yano, D., Mimura, S., Uda, K., and Suzuki, T.
  • Journal Title: Comp. Biochem. Physiol. Part B. Biochem. Mol. Biol. Volume: 198 Pages: 73-78
  • DOI: 10.1016/j.cbpb.2016.04.004
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Cooperativity and evolution of Tetrahymena two-domain arginine kinase. (2015)
  • Author(s): Okazaki, N., Motomura, S., Okazoe, N., Yano D. and Suzuki, T.
  • Journal Title: Int. J. Biol. Macromol. Volume: 79 Pages: 696-703
  • DOI: 10.1016/j.ijbiomac.2015.05.034
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Arginine kinases from the marine feather star Tropiometra afra macrodiscus: the first finding of a prenylation signal sequence in metazoan phosphagen kinases. (2015)
  • Author(s): Chouno, K., Yano, D., Uda, K., Fujita, T., Iwasaki, I. and Suzuki, T.
  • Journal Title: Comp. Biochem. Physiol. Part B. Biochem. Mol. Biol. Volume: 187 Pages: 55-64
  • DOI: 10.1016/j.cbpb.2015.04.014
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] ヨツヒメゾウリムシのアルギニンキナーゼ(AK)遺伝子のRNA干渉によるアルギニンリン酸シャトル機構の生理学的検証 (2017)
  • Author(s): 矢野大地、舟谷亮二、松岡達臣、鈴木知彦
  • Organizer: 日本動物学会第88回大会
• [Presentation] 原生生物におけるフォスファゲンキナーゼの多様性と酵素特性の解明 (2017)
  • Author(s): 矢野大地，舟谷亮二，松岡達臣，鈴木知彦
  • Organizer: 日本原生生物学会（つくば）
• [Presentation] Arginine kinase (AK) and arginine phosphate shuttle in Paramecium tetraurelia (2016)
  • Author(s): Daichi Yano, Tomohiko Suzuki
  • Organizer: The 22nd International Congress of Zoology and the 87th meeting of Zoological Science of Japan
  • Place of Presentation: 沖縄県宜野湾市
  • Int'l Joint Research
• [Presentation] ヨツヒメゾウリムシの繊毛運動におけるATP輸送とアルギニンリン酸シャトル機構 (2015)
  • Author(s): 矢野大地，鈴木知彦
  • Organizer: 日本動物学会第86回大会
  • Place of Presentation: 新潟市：新潟コンベンションセンター
  • Year and Date: 2015-09-19