Interaction of phomopsin A with porcine brain tubulin. Inhibition of tubulin polymerization and binding at a rhizoxin binding site.
Phomopsin A is an antimitotic cyclic peptide containing a 13-member ring including an ether linkage. It was isolated from the fungus Phomopsis leptostromiformis as the causal agent of lupinosis. Phomopsin A strongly inhibited microtubule assembly (IC50: 2.4 microM). Our study using radiolabeled phomopsin A, prepared biosynthetically by feeding L-[U-14C]isoleucine to the culture of P. leptostromiformis, indicated that at least two binding sites of phomopsin A exist on tubulin on the basis of a Scatchard analysis; i.e. the dissociation constants of a high affinity site (Kd1) and a low affinity site (Kd2) at 37 degrees were determined to be 1 x 10(-8) and 3 x 10(-7) M, respectively. Phomopsin A inhibited the binding of radiolabeled rhizoxin to tubulin with an inhibition constant (Ki) of 0.8 x 10(-8) M. This showed that the high affinity site of phomopsin A is identical to the rhizoxin binding site. The binding of the radiolabeled phomopsin A was also inhibited by rhizoxin and ansamitocin P-3, with an inhibition constant of 10(-7) M, and to a lesser extent by vinblastine. Phomopsin A had no inhibitory effect on colchicine binding to tubulin.
Li
Y
Y
Institute of Applied Microbiology, University of Tokyo, Japan.
Kobayashi
H
H
Tokiwa
Y
Y
Hashimoto
Y
Y
Iwasaki
S
S
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D003078:Colchicine / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D018942:Macrolides; D008453:Maytansine / Q000031:analogs & derivatives / Q000378:metabolism; D008868:Microtubule Proteins / Q000187:drug effects; D009183:Mycotoxins / Q000302:isolation & purification / Q000378:metabolism*; D011108:Polymers; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D050257:Tubulin Modulators; D014747:Vinblastine / Q000378:metabolism; D009842:Oligopeptides
D003078:Colchicine; D007783:Lactones; D018942:Macrolides; D008453:Maytansine; D008868:Microtubule Proteins; D009183:Mycotoxins; D011108:Polymers; D014404:Tubulin; D050257:Tubulin Modulators; D014747:Vinblastine; C035753:phomopsin; D009842:Oligopeptides; C015682:ansamitocins; C041443:rhizoxin