Radicicol binds and inhibits mammalian ATP citrate lyase.
Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase with K(i) values for citrate and ATP of 13 and 7 microm, respectively.
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Departments of Biotechnology and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Ishigami
K
K
Kitahara
T
T
Kasahara
K
K
Yoshida
M
M
Horinouchi
S
S
D001275:ATP Citrate (pro-S)-Lyase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D000818:Animals; D016227:Benzoquinones; D001667:Binding, Competitive; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000138:chemical synthesis* / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D018942:Macrolides; D011485:Protein Binding; D011809:Quinones / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology; D017828:Rifabutin / Q000031:analogs & derivatives
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; D001275:ATP Citrate (pro-S)-Lyase; C035359:monorden