Nitration of profilin effects its interaction with poly (L-proline) and actin.
Profilin from bovine spleen was nitrated with peroxynitrite; immunoblotting and spectrophotometric quantitation of nitrotyrosine residues suggested nitration of a single tyrosine residue in profilin with a stoichiometry of 0.6 mol of nitrotyrosine/mole of profilin. A decrease in the nitrotyrosine immunoreactivity of nitroprofilin during digestion with carboxypeptidase Y indicated that nitrotyrosine is located at the C-terminus of profilin. Nitroprofilin interaction with ligands such as phosphatidylinositol 4,5-bisphosphate, actin and poly (l-proline) was analyzed by monitoring the tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no significant difference in affinity of nitroprofilin to phosphatidylinositol 4,5-bisphosphate (K(d) of 4.8 +/- 0.5 muM for profilin, and K(d) of 5.7 +/- 0.6 muM for nitroprofilin), while poly (l-proline) binding studies revealed a twenty-fold increase in the affinity of profilin to poly (l-proline) upon nitration (K(d) of 21.8 +/- 1.7 muM for profilin, and K(d) of 1.1 +/- 0.1 muM for nitroprofilin). Actin polymerization studies involving pyrene-labeled actin indicated that profilin nitration inhibits the actin sequestering property of profilin. The critical actin monomer concentration (C(c)) was 150 and 250 nM in the presence of nitroprofilin and profilin, respectively. Thus, nitric oxide and free radicals produced under different conditions could alter the functions of profilin through nitration, such as its interaction with actin and poly (l-proline).
Kasina
S
S
Hansen's Life Sciences Research Building, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47906, USA.
Rizwani
Wasia
W
Radhika
K V N
KV
Singh
Surya S
SS
D000199:Actins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D030421:Peroxynitrous Acid; D051304:Profilins / Q000737:chemistry* / Q000378:metabolism; D013154:Spleen; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000199:Actins; D009566:Nitrates; D010455:Peptides; D051304:Profilins; D030421:Peroxynitrous Acid; C011083:polyproline; C002744:3-nitrotyrosine; D014443:Tyrosine