Key result
Interaction of PRODAN-F-actin with DNAase I, a 19 kDa protein, and cytochalasin D reduced emission intensity and caused a red shift, whereas myosin, tropomyosin, and phalloidin had no effect.
The study demonstrates how different actin-binding proteins and drugs alter the fluorescence emission of PRODAN-labelled actin, providing insights into actin filament structure and interactions.
Supports PRODAN-F-actin as an in vitro probe for select actin interactions; leaves open translation to cellular or clinical settings.
The influence of various actin-binding proteins and drugs on the fluorescence emission of rabbit muscle actin labelled with the fluorescent probe acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at Cys-374, the penultimate amino acid residue of the actin amino acid sequence, was studied. Addition of myosin, tropomyosin or phalloidin, agents known to bind only to filamentous F-actin, did not change the emission energy or the integrated intensity of the fluorescence spectrum. The presence of heavy meromyosin or of the glycolytic enzyme aldolase led to a small (approx. 2%) increase in the integrated intensity, and in the energy of the emitted fluorescence. The interaction of 6-propionyl-2-(NN-dimethyl)aminonaphthalene (PRODAN)-F-actin with pancreatic DNAase I and with a filament-severing 19 kDa protein from pig brain resulted in the gradual reduction of the integrated intensity of the emission and a red shift of the emission energy, suggestive of a disintegration of the actin filament structure. Profilin caused a < 10% change in the emission energy. Cytochalasin D reduced the integrated intensity of PRODAN-F-actin and red-shifted the emission energy, while cytochalasin B was without influence. Pancreatic DNAase I did not change the fluorescence emission of PRODAN-G-actin, suggesting that binding of this enzyme does not alter the environment of the probe. When the 19 kDa protein bound to PRODAN-G-actin, however, the integrated intensity was reduced and the emission energy was lowered. This effect was exploited to estimate the binding constant for the interaction between the 19 kDa protein and PRODAN-G-actin. The Kd was found to be about 0.25 microM.
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Kasper Zechel (1993) studied this question. Actin-binding proteins and drugs was evaluated on Fluorescence emission energy and integrated intensity. Interaction of PRODAN-F-actin with DNAase I, a 19 kDa protein, and cytochalasin D reduced emission intensity and caused a red shift, whereas myosin, tropomyosin, and phalloidin had no effect.
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