Actin-binding specificity of marine macrolide toxins, mycalolide B and kabiramide D.
An actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actin-depolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.
Wada
S
S
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Matsunaga
S
S
Saito
S
S
Fusetani
N
N
Watabe
S
S
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001704:Biopolymers; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008387:Marine Toxins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D010080:Oxazoles / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D013050:Spectrometry, Fluorescence
D000199:Actins; D001704:Biopolymers; D008387:Marine Toxins; D008840:Microfilament Proteins; D010080:Oxazoles; C112787:kabiramide D; C080978:mycalolide B; C013411:biocytin; D008239:Lysine