A continuous fluorometric assay for phospholipase A(2) activity in brain cytosol.
Alterations in phospholipase A(2) (PLA(2)) activity have been implicated in Alzheimer disease and other neurological disorders, although brain PLA(2) activity is currently measured using lengthy, non-continuous assays. We describe herein a rapid, continuous assay in which we measured PLA(2) activity in mouse brain cytosol (CB-57). Brains were homogenized in HEPES buffer (pH 7.5) and the cytosolic fraction was prepared by centrifugation at 25000xg for 20 min, followed by centrifugation of the supernatant at 100000xg for 60 min. Cytosolic protein content was determined using the Bradford assay. Pyrene labeled phosphatidylcholine was added to 50 microg of cytosolic protein in Tris buffer (pH 8.0) containing fatty acid free-bovine serum albumin for a final assay volume of 2 ml. Assay temperature was maintained at 30+/-1 degrees C. The excitation wavelength was 345 nm and emission was measured at 377 nm. Fluorescence intensity was converted to molar concentrations using a standard curve. Under these conditions, bromoenol lactone inhibited up to 58% of the PLA(2) activity with an IC(50) of 0.5 microM. In a separate experiment, lack of appreciable alternative acylhydrolase activity was verified chromatographically. Using this method, brain PLA(2) activity can be measured in a continuous, rapid, and sensitive manner.
Yarger
D E
DE
Department of Biology, Cumberland College, Williamsburg, KY 40769, USA.
Patrick
C B
CB
Rapoport
S I
SI
Murphy
E J
EJ
D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology*; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D003080:Cold Temperature / Q000009:adverse effects; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D005260:Female; D005470:Fluorometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D009281:Naphthalenes / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis* / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D002457:Cell Extracts; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; D011753:Pyrones; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D010741:Phospholipases A