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Anal Biochem. 2018 May 15;549:164-170. doi: 10.1016/j.ab.2018.03.024. Epub 2018 Mar 30.

A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid.

Author information

1
Department of Ophthalmology, School of Medicine, Sapporo Medical University, Sapporo, Japan. Electronic address: abeakira@sapmed.ac.jp.
2
Department of Ophthalmology, School of Medicine, Sapporo Medical University, Sapporo, Japan.
3
Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI, USA.

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 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.

KEYWORDS:

Fluorescence-labeled oxidized phospholipid; Lysosomal phospholipase A2; Truncated oxidized phospholipid; sn-1 preference

PMID:
29605449
DOI:
10.1016/j.ab.2018.03.024
[Indexed for MEDLINE]

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