Modification of gelsolin with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole.
Pig plasma gelsolin was modified with the fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) for lysyl residues. The relationship between the gelsolin activity and the degree of NBD labeling suggested that a single lysyl residue, which reacted five times slower than the other reactive lysyl residues, was essential for the activity. Taking advantage of the slow reactivity of the essential residue, active NBD-gelsolin was prepared. Limited cleavage of NBD-gelsolin by chymotrypsin indicated that the fluorescent reagents were randomly incorporated into all fragments observed. When NBD-gelsolin formed a gelsolin/actin (1:2) complex in the presence of micromolar Ca2+, the fluorescence spectra of NBD-gelsolin were red-shifted by 5 nm and the intensity decreased by 30%. However, on binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), the fluorescence spectra were blue-shifted by 5 nm with a concomitant increase in intensity by 20%. The addition of PtdIns(4,5)P2 to the NBD-gelsolin actin (1:2) complex restored the fluorescence spectra to that obtained in the presence of PtdIns(4,5)P2 alone. These results indicated that NBD-gelsolin, selectively labeled on lysyl residues not essential for activity, can be a useful probe to monitor the binding of PtdIns(4,5)P2 and actin.
Doi
Y
Y
Department of Food Science, Kyoto Women's University, Japan.
Hashimoto
T
T
Yamaguchi
H
H
Vertut-Doï
A
A
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000302:isolation & purification; D002918:Chymotrypsin; D005456:Fluorescent Dyes; D018260:Gelsolin; D007700:Kinetics; D008840:Microfilament Proteins / Q000097:blood* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013552:Swine
D000199:Actins; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D002918:Chymotrypsin; D009327:4-Chloro-7-nitrobenzofurazan