| Title : A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid - Abe_2018_Anal.Biochem_549_164 |
| Author(s) : Abe A , Hiraoka M , Shayman JA , Ohguro H |
| Ref : Analytical Biochemistry , 549 :164 , 2018 |
| Abstract : 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 the sn-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. The Km and Vmax values of the recombinant LPLA2 for PGPE-BODIPY were 5.64 microM and 20.7 micromol/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. |
| ESTHER : Abe_2018_Anal.Biochem_549_164 |
| PubMedSearch : Abe_2018_Anal.Biochem_549_164 |
| PubMedID: 29605449 |
Abe A, Hiraoka M, Shayman JA, Ohguro H (2018)
A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid
Analytical Biochemistry
549 :164
Abe A, Hiraoka M, Shayman JA, Ohguro H (2018)
Analytical Biochemistry
549 :164