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Eur J Biochem ; 263(1): 85-92, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10429191

RESUMO

Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin.


Assuntos
Actinas/metabolismo , Proteínas Contráteis , Corantes Fluorescentes/metabolismo , Gelsolina/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfatidilinositóis/metabolismo , 4-Cloro-7-nitrobenzofurazano , Fatores de Despolimerização de Actina , Animais , Sítios de Ligação , Bovinos , Corantes Fluorescentes/química , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/metabolismo , Lipossomos , Micelas , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 4,5-Difosfato/análogos & derivados , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/química , Profilinas , Ligação Proteica , Pirenos , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Proteínas tau/metabolismo
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