The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes

被引:29
作者
Bolean, M. [1 ]
Simao, A. M. S. [1 ]
Favarin, B. Z. [1 ]
Millan, J. L. [2 ]
Ciancaglini, P. [1 ]
机构
[1] Univ Sao Paulo, Dept Quim, FFCLRP USP, BR-14040901 Ribeirao Preto, SP, Brazil
[2] Sanford Burnham Med Res Inst, Sanford Childrens Hlth Res Ctr, La Jolla, CA USA
基金
巴西圣保罗研究基金会;
关键词
Matrix vesicles; GPI-anchored; Alkaline phosphatase; Cholesterol; Biomimetic system; Differential scanning calorimetry; THERMOTROPIC PHASE-BEHAVIOR; MATRIX VESICLE CALCIFICATION; GROWTH-PLATE; LIPID RAFTS; PHOSPHOLIPASE-C; BONE; PHOSPHATIDYLINOSITOL; MEMBRANES; BILAYER; CELLS;
D O I
10.1016/j.bpc.2010.08.002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (T-c) and enthalpy variation (Delta H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a T-c of 41.5 degrees C and Delta H of 7.63 Kcal mol(-1). The gradual increase in cholesterol concentration decrease Delta H values, reaching a Delta H of 0.87 Kcal mol(-1) for DPPC: cholesterol system with 36 mol% of cholesterol. An increase in T-c, up to 47 degrees C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in Delta H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification. (C) 2010 Elsevier B.V. All rights reserved.
引用
收藏
页码:74 / 79
页数:6
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