Caseli Luciano, Oliveira Rafael G, Masui Douglas C, Furriel Rosa P M, Leone Francisco A, Maggio Bruno, Zaniquelli M Elisabete D
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Langmuir. 2005 Apr 26;21(9):4090-5. doi: 10.1021/la047292s.
The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.
已在不同表面压力下的朗缪尔磷脂单分子层中研究了糖基磷脂酰肌醇（GPI）锚定碱性磷酸酶的催化活性。将酶底物对硝基苯磷酸注入混合酶/脂质朗缪尔单分子层的亚相中。通过监测朗缪尔槽单分子层亚相中410nm处的吸光度来跟踪其水解产物。通过单分子层的横向压缩获得了对应于不同分子表面密度的几种表面压力。通过荧光显微镜观察到的单分子层形态显示，由于酶在混合酶/脂质膜内的不均匀分配，存在三种不同类型的区域。催化活性受酶表面密度调节，在达到18mN/m的压力之前活性增加，但当平衡面内弹性（表面压缩模量）更明显增加时活性显著降低，导致界面形态发生变化。提出了一个通过气/液界面处的脂质/酶表面形态和酶表面堆积来调节酶取向和催化活性的模型。这些结果可能对理解生物膜中GPI锚定蛋白的酶活性调节具有重要影响。
