A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid.
A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid.
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DOI：
10.1016/j.ab.2018.03.024
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发表时间：
2018-05
影响因子：
2.9
通讯作者：
A. Abe;M. Hiraoka;J. Shayman;H. Ohguro
中科院分区：
文献类型：
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作者：
A. Abe;M. Hiraoka;J. Shayman;H. Ohguro
溶酶体磷脂酶A2（LPLA 2）是参与细胞磷脂稳态的关键酶。最近，LPLA 2被报道优先降解一些截短的氧化磷脂在thesn-1位置。使用市售的截短氧化磷脂与荧光染料1-棕榈酰-2-戊二酰-sn-甘油基-3-磷酸乙醇胺-N-[4-（二吡咯亚甲基二氟化硼）丁酰基]（PGPE-BODIPY）结合，以开发该酶的特异性测定。当重组小鼠LPLA 2与由1，2-O-十八烷基-sn-甘油-3-磷酸胆碱/PGPE-BODIPY组成的脂质体在酸性条件下孵育时，PGPE-BODIPY转化为棕榈酸和极性BODIPY产物。在通过氯仿/甲醇进行相分配后，在水相中回收极性BODIPY产物，并鉴定为1-溶血-PGPE-BODIPY。通过荧光测量定量测定1-lyso-PGPE-BODIPY的形成。重组LPLA 2对PGPE-BODIPY的Km和Vmax分别为5.64 μM和20.7 μmol/min/mg蛋白。在LPLA 2缺陷小鼠血清中存在对PGPE-BODIPY的可检测活性，但用4-（2-氨乙基）苯磺酰氟（AEBSF）处理完全抑制了脱酰酶活性。AEBSF对LPLA 2活性无影响。用该方法对AEBSF预处理的小鼠血清LPLA 2活性进行了特异性定量测定。基于PGPE-BODIPY和AEBSF的LPLA 2测定是方便的，并且可用于测量各种生物样本中的LPLA 2活性。
Lysosomal phospholipase A2 (LPLA2) is a key enzyme involved in the homeostasis of cellular phospholipids. Recently, LPLA2 was reported to preferentially degrade some truncated oxidized phospholipids at thesn-1 position. A commercially available, truncated oxidized phospholipid conjugated with a fluorescent dye, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine-N-[4-(dipyrrometheneboron difluoride) butanoyl] (PGPE-BODIPY), was used to develop a specific assay for this enzyme. When recombinant mouse LPLA2 was incubated with liposomes consisting of 1,2-O-octadecyl-sn-glycero-3-phosphocholine/PGPE-BODIPY under acidic conditions, PGPE-BODIPY was converted to palmitic acid and a polar BODIPY-product. After phase partitioning by chloroform/methanol, the polar BODIPY-product was recovered in the aqueous phase and identified as 1-lyso-PGPE-BODIPY. The formation of 1-lyso-PGPE-BODIPY was quantitatively determined by fluorescent measurements. TheKmandVmaxvalues of the recombinant LPLA2 for PGPE-BODIPY were 5.64 μM and 20.7 μmol/min/mg protein, respectively. Detectable activity against PGPE-BODIPY was present in LPLA2 deficient mouse sera, but the deacylase activity was completely suppressed by treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF had no effect on LPLA2 activity. The LPLA2 activity of mouse serum pre-treated with AEBSF was specifically and quantitatively determined by this assay method. The PGPE-BODIPY and AEBSF based LPLA2 assay is convenient and can be used to measure LPLA2 activity in a variety of biological specimens.