Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin.
Reaction between horse plasma gelsolin and fluorescein-5-isothiocyanate (FITC) resulted in incorporation of 4.8 +/- 0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with alpha-chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC-gelsolin has a peptide-backbone circular dichroism spectrum at 20 degrees C that is indistinguishable from that of native gelsolin, but FITC-gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC-gelsolin is fully able to carry out severing of F-actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC-gelsolin to quenching by I-. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC-gelsolin solutions. A Ca(2+)-sensitive interaction between gelsolin and tropomyosin also is observed.
Department of Chemistry, University of British Columbia, Vancouver, Canada.
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Koepf
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Burtnick
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000737:chemistry; D002942:Circular Dichroism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D018260:Gelsolin; D006736:Horses; D008239:Lysine; D008840:Microfilament Proteins / Q000097:blood* / Q000737:chemistry; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D014335:Tropomyosin; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine; D002118:Calcium