A rapid assay for activity of phospholipase A2 using radioactive substrate
| Autor: | Masuyuki Katsumata, Chhanda Gupta, Allen S. Goldman |
|---|---|
| Rok vydání: | 1986 |
| Předmět: |
Octoxynol
Silicic Acid Palmitic Acid Biophysics 1-Propanol Palmitic Acids Tritium Biochemistry Phospholipases A Polyethylene Glycols Substrate Specificity Palmitic acid chemistry.chemical_compound Acetic acid Phospholipase A2 Phosphatidylcholine Sodium sulfate Hexanes Bovine serum albumin Molecular Biology Chromatography biology Water Substrate (chemistry) Pulmonary Surfactants Cell Biology Enzyme assay Phospholipases A2 chemistry Phospholipases Isotope Labeling biology.protein Chromatography Thin Layer Deoxycholic Acid |
| Zdroj: | Analytical Biochemistry. 154:676-681 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/0003-2697(86)90046-1 |
| Popis: | A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, l -α-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 m m ) is dissolved in 80% ethanol containing 25 m m sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 m glycine-NaOH buffer, pH 9.0, containing 2 μmol CaCl2, 10 μg bovine serum albumin, 2.5 μmol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40–100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 μmol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, l -α-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC. |
| Databáze: | OpenAIRE |
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