1992 Fiscal Year Annual Research Report

アカパンカビのATP-GTP結合蛋白質を含む信号伝達系遺伝子の解析

Research Project

Project/Area Number	02454002
Research Institution	Yokohama City University, Kihara Institute for Biological Research
Principal Investigator	蓮沼仰嗣横浜市立大学, 木原生物学研究所, 教授 (80012475)
Keywords	アカパンカビ / 信号伝達 / Ras蛋白質 / 光信号受容 / UV-A光 / 青色光 / wc-1、wc-2 / F_0F_1-ATPase
Research Abstract	アカパンカビ膜分画に多数のNTP結合蛋白質を見い出した。Ras特異抗体を用いて、21kDa、24kDa、26kDa、27kDa蛋白質をウエスタンブロットにより検出した。そのうち26kDa蛋白質を部分精製した。26kDa蛋白質はATP、GTP、CTP、UTPの結合能を持ち、[γ-^<32>P]ATP、[γ-^<32>P]GTPからの自己リン酸化がなされた。 v-Ha-rasをプローブにし、ras関連遺伝子NC-ras2A、NC-ras2Bをクローニングした。NC-ras2AについてはcDNAもクローン化され、塩基配列も決定された。NC-ras2BはNC-ras2Aに相似するが異なる遺伝子であり、部分的に塩基配列が決定された。これらの遺伝子はras蛋白質のもつ全ての共通配列を含んでいた。菌糸の膜分画を用いてUV-A光(355nm)1秒照射によりATP、GTPを結合する58kDa、77kDa、83kDa、129kDa蛋白質を見い出した。83kDa蛋白質へのGTPの結合はトランスデューシンC末特異抗体で、UV-A光の信号が入らなくなった。トランスデューシン-1及び-2のC末mRNAを用いて、アカパンカビのゲノムライブラリーより5つのクローンを単離した。そのうち1つはNC-ras2Aにも強い相同性を示した。このクローンの塩基配列決定を進めている。さらに膜分画を用いて、青色光(465nm)1秒照射により、0℃5秒でリン酸化の促進される15kDa蛋白質が見い出された。光に感受性の変異株であるwc-1、wc-2変異株では、この15kDa蛋白質のリン酸化が見られなかった。しかし両者の膜分画を混合すると光信号によるリン酸化の上昇が見られた。これらのリン酸化はATPaseの阻害剤である。DCCD、DES、オリゴマイシンにより、光信号の入りが阻害された。さらに15kDa蛋白質のリン酸化と共役して70kDa蛋白質のリン酸化が存在する可能性が示唆されている。70kDa蛋白質のリン酸化はアルカリ性に耐性で、ヒスチジンのリン酸化と考えられる。これは大腸菌のCheA(73kDa)、cheY(15kDa)と相似しており、F_0F_1-ATPaseとの関連において今後解決されていかねばならない問題である。

Research Products
(19 results)

All Other

All Publications (19 results)

[Publications] Hasunuma,K.and Nishikawa,K.: "An NTP-binding protein that cross-reacts with a Ras-specific antibody in the mycelia of Neurospora crassa." Plant Cell Physiol.33. 889-896 (1992)
[Publications] Hasunuma,K.,Oda,K.and Nishikawa,K.: "Assay method and partial characterization of nucleoside triphosphate (NTP)-binding proteins in Neurospora crassa." Seiken Ziho.37. 1-6 (1992)
[Publications] Hasumura,K.: "NTP-binding proteins and endogenous ADP-ribosyl transferase in the soluble and membrane fractions of the mycelia of Neurospora crassa." Seiken Ziho.37. 7-18 (1992)
[Publications] Hasunuma,K.,Ootaki,T.and Suzuki,T.: "ATP-GTP(NTP)-binding proteins in the mycelia of Phycomyces blakesleeanus." Seiken Ziho.37. 19-30 (1992)
[Publications] Hasunuma,K.,Furukawa,K.and Kajiura,H.: "Partical amino acid sequence analysis of Pi repressible cyclic phosphodiesterase in Neurospora." Seiken Ziho.37. 31-36 (1992)
[Publications] Ito,K.and Hasunuma,K.: "Nucleotide specificites of NTP-binding proteins in the microsomal fraction from stem sections of etiolated pea seedlings." Seiken Ziho.37. 37-45 (1992)
[Publications] Hasunuma,K.: "Triton X-100 and SDS-polyacrylamide gel electrophoresis analysis of NTP-binding proteins in the plasma from the stem sections of etiolated pea seedlings." Seiken Ziho.37. 46-54 (1992)
[Publications] Hasunuma,K.,Oda,K.,Nishikawa,K.and Hamada,T.: "ATP-GTP(NTP)-binding proteins and light signal perception in a fungus and a plant." Yokohama Medical Bulletin. (1992)
[Publications] Hasunuma,k.,Oda,K.and Hamada,T.: "Binding of [α-^<32>P]NTP to cholera toxin and light signal transmission stimulating the binding of [α-^<32>P]GTP via pea plasma membrane." Cytologia.(1993)
[Publications] Hasunuma,K.and Oda,K.: "[α-^<32>P]NTP binding to diphtheria toxin and light signal transmission to the toxin through a microsomal fraction of Neurospora crassa." Cytologia.(1993)
[Publications] Hasunuma,K.and Hamada,T.: "ATP-GTP(NTP)-binding proteins and light signal transmission in the plasma membrane from etiolated pea seedlings." Cytologia.(1993)
[Publications] Hasunuma,K.,Ito,K.and Oda,K.: "Nucleoside Triphosphate(NTP)binding to pertussis toxin and light signal transmission to it through a microsomal fraction of the stem section of etiolated pea seedlings." Cytologia.(1993)
[Publications] Yoshida,S.,Kanauchi,A.,Hasunuma,K.and Murayama,T.: "Cloning and sequencing of a ras gene in Neurospora crassa." J.Tech.Res.37-1. (1993)
[Publications] Hasunuma,K.and Oda,K.: "Light Signal transmission binding [α-^<32>P]NTP to toxins through a membrane fraction of Neurospora." 11th Intern.Congr.Photobiol.Abstr.-343 (1992)
[Publications] Hasunuma,K.,Hamada,T.and Itoh,K.: "NTP-binding proteins and light signal perception in Pisum." 11th Intern.Congr.Photobiol.Abstr.-345 (1992)
[Publications] Oda,K.and Hasunuma,K.: "Light signal perception by NTP-binding proteins in Neurospora." 11th Intern.Congr.Photobiol.Abstr.-227 (1992)
[Publications] Hasegawa,K.,Shinozawa,T.,Hashimoto,H.,Hasunuma,K.,Shimamoto,M.and Tsukahara,Y.: "Photo pigments of the primordial visual system." 11th Intern.Congr.Photobiol.Abstr.-353 (1992)
[Publications] Oda,K.and Hasunuma,K.: "In vitro analysis of blue light signal perception in Neurospora crassa." Symp.“Molecular mechanism of the photo-reception and transduction in the bio-information systems" Tokyo.Abstr.-9 (1992)
[Publications] 蓮沼仰嗣: "光信号伝達の分子機構とリズム" 基礎老化研究, (1993)

1992 Fiscal Year Annual Research Report

アカパンカビのATP-GTP結合蛋白質を含む信号伝達系遺伝子の解析

Principal Investigator

蓮沼 仰嗣 横浜市立大学, 木原生物学研究所, 教授 (80012475)

Research Products

[Publications] Hasunuma,K.and Nishikawa,K.: "An NTP-binding protein that cross-reacts with a Ras-specific antibody in the mycelia of Neurospora crassa." Plant Cell Physiol.33. 889-896 (1992)

[Publications] Hasunuma,K.,Oda,K.and Nishikawa,K.: "Assay method and partial characterization of nucleoside triphosphate (NTP)-binding proteins in Neurospora crassa." Seiken Ziho.37. 1-6 (1992)

[Publications] Hasumura,K.: "NTP-binding proteins and endogenous ADP-ribosyl transferase in the soluble and membrane fractions of the mycelia of Neurospora crassa." Seiken Ziho.37. 7-18 (1992)

[Publications] Hasunuma,K.,Ootaki,T.and Suzuki,T.: "ATP-GTP(NTP)-binding proteins in the mycelia of Phycomyces blakesleeanus." Seiken Ziho.37. 19-30 (1992)

[Publications] Hasunuma,K.,Furukawa,K.and Kajiura,H.: "Partical amino acid sequence analysis of Pi repressible cyclic phosphodiesterase in Neurospora." Seiken Ziho.37. 31-36 (1992)

[Publications] Ito,K.and Hasunuma,K.: "Nucleotide specificites of NTP-binding proteins in the microsomal fraction from stem sections of etiolated pea seedlings." Seiken Ziho.37. 37-45 (1992)

[Publications] Hasunuma,K.: "Triton X-100 and SDS-polyacrylamide gel electrophoresis analysis of NTP-binding proteins in the plasma from the stem sections of etiolated pea seedlings." Seiken Ziho.37. 46-54 (1992)

[Publications] Hasunuma,K.,Oda,K.,Nishikawa,K.and Hamada,T.: "ATP-GTP(NTP)-binding proteins and light signal perception in a fungus and a plant." Yokohama Medical Bulletin. (1992)

[Publications] Hasunuma,k.,Oda,K.and Hamada,T.: "Binding of [α-^<32>P]NTP to cholera toxin and light signal transmission stimulating the binding of [α-^<32>P]GTP via pea plasma membrane." Cytologia.(1993)

[Publications] Hasunuma,K.and Oda,K.: "[α-^<32>P]NTP binding to diphtheria toxin and light signal transmission to the toxin through a microsomal fraction of Neurospora crassa." Cytologia.(1993)

[Publications] Hasunuma,K.and Hamada,T.: "ATP-GTP(NTP)-binding proteins and light signal transmission in the plasma membrane from etiolated pea seedlings." Cytologia.(1993)

[Publications] Hasunuma,K.,Ito,K.and Oda,K.: "Nucleoside Triphosphate(NTP)binding to pertussis toxin and light signal transmission to it through a microsomal fraction of the stem section of etiolated pea seedlings." Cytologia.(1993)

[Publications] Yoshida,S.,Kanauchi,A.,Hasunuma,K.and Murayama,T.: "Cloning and sequencing of a ras gene in Neurospora crassa." J.Tech.Res.37-1. (1993)

[Publications] Hasunuma,K.and Oda,K.: "Light Signal transmission binding [α-^<32>P]NTP to toxins through a membrane fraction of Neurospora." 11th Intern.Congr.Photobiol.Abstr.-343 (1992)

[Publications] Hasunuma,K.,Hamada,T.and Itoh,K.: "NTP-binding proteins and light signal perception in Pisum." 11th Intern.Congr.Photobiol.Abstr.-345 (1992)

[Publications] Oda,K.and Hasunuma,K.: "Light signal perception by NTP-binding proteins in Neurospora." 11th Intern.Congr.Photobiol.Abstr.-227 (1992)

[Publications] Hasegawa,K.,Shinozawa,T.,Hashimoto,H.,Hasunuma,K.,Shimamoto,M.and Tsukahara,Y.: "Photo pigments of the primordial visual system." 11th Intern.Congr.Photobiol.Abstr.-353 (1992)

[Publications] Oda,K.and Hasunuma,K.: "In vitro analysis of blue light signal perception in Neurospora crassa." Symp.“Molecular mechanism of the photo-reception and transduction in the bio-information systems" Tokyo.Abstr.-9 (1992)

[Publications] 蓮沼 仰嗣: "光信号伝達の分子機構とリズム" 基礎老化研究, (1993)

蓮沼仰嗣横浜市立大学, 木原生物学研究所, 教授 (80012475)

[Publications] 蓮沼仰嗣: "光信号伝達の分子機構とリズム" 基礎老化研究, (1993)