TY - JOUR
T1 - Detection of Rap1A as a yessotoxin binding protein from blood cell membranes
AU - Ujihara, Satoru
AU - Oishi, Tohru
AU - Mouri, Ryota
AU - Tamate, Rie
AU - Konoki, Keiichi
AU - Matsumori, Nobuaki
AU - Murata, Michio
AU - Oshima, Yasukatsu
AU - Sugiyama, Naoyuki
AU - Tomita, Masaru
AU - Ishihama, Yasushi
N1 - Funding Information:
We are grateful to Professor Toshifumi Takao for discussion and helps in pretreatment of protein samples for nano LC–MS/MS experiments. This work was supported by Grants-in-Aid for Scientific Research (S) (No. 18101010 ), and (B) (No. 21350028 ) from MEXT, Japan, and The Naito Foundation. Research fellowships for Young Scientists to S.U. from JSPS, and from GCOE program, ‘Global Education and Research Center for Bio-Environmental Chemistry, Osaka University’ are acknowledged.
PY - 2010/11/15
Y1 - 2010/11/15
N2 - As is the case with other ladder-shaped polyether compounds, yessotoxin is produced by marine dinoflagellate, and possesses various biological activities beside potent toxicity. To gain a better understanding of the molecular mechanism for high affinity between these polyethers and their binding proteins, which accounts for their powerful biological activities, we searched for its binding proteins from human blood cells by using the biotin-conjugate of desulfated YTX as a ligand. By a protein pull-down protocol with use of streptavidin beads, a band of specifically binding proteins was detected in SDS-PAGE. HPLC-tandem mass spectrometry (MS/MS) indicated that Rap 1A, one of Ras superfamily proteins, binds to the YTX-linked resins. Western blotting and surface plasmon resonance experiments further confirmed that Rap1A specifically binds to YTX with the KD value around 4 μM.
AB - As is the case with other ladder-shaped polyether compounds, yessotoxin is produced by marine dinoflagellate, and possesses various biological activities beside potent toxicity. To gain a better understanding of the molecular mechanism for high affinity between these polyethers and their binding proteins, which accounts for their powerful biological activities, we searched for its binding proteins from human blood cells by using the biotin-conjugate of desulfated YTX as a ligand. By a protein pull-down protocol with use of streptavidin beads, a band of specifically binding proteins was detected in SDS-PAGE. HPLC-tandem mass spectrometry (MS/MS) indicated that Rap 1A, one of Ras superfamily proteins, binds to the YTX-linked resins. Western blotting and surface plasmon resonance experiments further confirmed that Rap1A specifically binds to YTX with the KD value around 4 μM.
KW - Ladder-shaped polyether
KW - Pull-down assay
KW - Ras superfamily proteins
KW - Yessotoxin
KW - ap1A
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U2 - 10.1016/j.bmcl.2010.09.080
DO - 10.1016/j.bmcl.2010.09.080
M3 - Article
C2 - 20943388
AN - SCOPUS:77958037739
VL - 20
SP - 6443
EP - 6446
JO - Bioorganic and Medicinal Chemistry Letters
JF - Bioorganic and Medicinal Chemistry Letters
SN - 0960-894X
IS - 22
ER -
