Phalloidin unzips nebulin from thin filaments in skeletal myofibrils.
Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofibrils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incubation of skeletal myofibrils with Ca(2+)-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incorporated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin 'unzips' nebulin starting from the pointed ends of the thin filaments.
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Boston Biomedical Research Institute, MA 02114, USA.
Lehrer
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D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism; D000939:Epitopes / Q000502:physiology; D005455:Fluorescent Antibody Technique; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000276:immunology / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology*; D011485:Protein Binding / Q000502:physiology; D011817:Rabbits; D012235:Rhodamines; D012518:Sarcomeres / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D003374:Coumarins; D000939:Epitopes; D009124:Muscle Proteins; D012235:Rhodamines; C035620:nebulin; D010590:Phalloidine; C030123:coumarin; D002118:Calcium