The substrate specificities of four different lysophospholipases as determined by a novel fluorescence assay.
A novel fluorescence assay for quantifying lysophospholipase activity is described which utilizes a commercially available acrylodated intestinal fatty-acid-binding protein (ADIFAB) and non-radiolabelled substrate. Quantification of enzyme activity is based on the decrease in ADIFAB fluorescence at 432 nm in the presence of nanomolar concentrations of non-esterified ('free') fatty acids. Lysophospholipase activity measured by the ADIFAB assay and a conventional radiometric assay yield comparable results and have comparable levels of sensitivity (approximately 10 pmol/min per ml). The ADIFAB assay has the advantageous features of continuous monitoring of enzyme activity and the availability of a broad range of potential substrates, because non-radiolabelled lysophospholipids can be employed in the assay. The hydrolytic activities of four lysophospholipases were determined, including a bacterial secreted phospholipase A2/lysophospholipase, the human-eosinophil-secreted lysophospholipase, a human intracellular lysophospholipase (peak 3) isolated from HL-60 cells and a high-molecular-mass cytosolic phospholipase A2/lysophospholipase from a mouse mammary carcinoma. Each of these enzymes was found to have a distinctive hydrolytic profile as determined by an array of lysophospholipids differing in their polar headgroups and sn-1 fatty-acyl substituents.
She
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Schering Plough Research Institute, Kenilworth, NJ 07033.
Garsetti
D E
DE
Steiner
M R
MR
Egan
R W
RW
Clark
M A
MA
D002352:Carrier Proteins; D002460:Cell Line; D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008245:Lysophospholipase / Q000378:metabolism*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011994:Recombinant Proteins; C079339:acrylodated intestinal fatty acid binding protein, recombinant; D008245:Lysophospholipase