The interaction of 6-propionyl-2-(NN-dimethyl)aminonaphthalene (PRODAN)-labelled actin with actin-binding proteins and drugs.
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.
Zechel
K
K
Max-Planck-Institut for Biophysical Chemistry, Department of Biochemistry, Göttingen, Federal Republic of Germany.
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D003571:Cytochalasin B / Q000737:chemistry; D015638:Cytochalasin D / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D010590:Phalloidine / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011817:Rabbits; D013552:Swine
D000199:Actins; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D004364:Pharmaceutical Preparations; D010590:Phalloidine; D015638:Cytochalasin D; D003571:Cytochalasin B; C038517:prodan; D015081:2-Naphthylamine; D003545:Cysteine