Tuominen E K, Holopainen J M, Chen J, Prestwich G D, Bachiller P R, Kinnunen P K, Janmey P A
Lipid Research Laboratory, Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, Finland.
Eur J Biochem. 1999 Jul;263(1):85-92. doi: 10.1046/j.1432-1327.1999.00464.x.
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.
合成了磷脂酰肌醇（PtdIns）-(4,5)-P2的荧光衍生物，并用于测试PtdIns-(4,5)-P2调节蛋白凝溶胶蛋白、tau蛋白、丝切蛋白和前纤维蛋白对以胶束形式存在或与磷脂酰胆碱（PtdCho）混合于双层囊泡中的标记PtdIns-(4,5)-P2的影响。凝溶胶蛋白增加了7-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯（NBD）或芘标记的PtdIns-(4,5)-P2以及NBD-PtdIns-(3,4,5)-P3的荧光。丝切蛋白和前纤维蛋白与PtdIns-(4,5)-P2等摩尔比时未产生可检测到的变化，而tau蛋白降低了NBD-PtdIns-(4,5)-P2的荧光。凝溶胶蛋白对混合脂质囊泡中NBD-PtdIns-(4,5)-P2的荧光增强取决于双层中PtdIns-(4,5)-P2的摩尔分数。3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho的特异性增强远低于10% PtdIns-(4,5)-P2 : 90% PtdCho，但添加7%未标记的PtdIns-(4,5)-P2可增加3% NBD-PtdIns-(4,5)-P2的增强效果。添加Ca2+或G-肌动蛋白可逆转凝溶胶蛋白依赖性的NBD-PtdIns-(4,5)-P2荧光增加。凝溶胶蛋白N端结构域（第1 - 160位氨基酸残基）和由凝溶胶蛋白第150 - 169位氨基酸残基组成的肽段观察到显著但较弱的荧光增强。完整的凝溶胶蛋白与芘-PtdIns-(4,5)-P2之间的荧光能量转移比含有PtdIns-(4,5)-P2结合位点的凝溶胶蛋白N端区域要强得多，这表明PtdIns-(4,5)-P2可能结合在凝溶胶蛋白N端和C端结构域并列形成的位点附近。
