| Autor: |
Bolognese, B. J., Holmes, S. D., McMillan, L. J., Kaiser, K. F., Marshall, L. A. |
| Zdroj: |
Inflammopharmacology; September 1997, Vol. 5 Issue: 3 p247-260, 14p |
| Abstrakt: |
The metabolism of arachidonic acid into inflammatory mediators (e.g. prostaglandin, leukotrienes) is dependent upon the rate-limiting enzyme phospholipase A2. Localization and quantification of type II 14 kDa phospholipase A2(PLA2) in cells or tissue preparations has historically been accomplished through activity measurements, a process that can provide variable results due to interference by exogenous substances with hydrolysis assessment. Others have reported on the use of sandwich enzyme immunoassays (EIA) to measure 14 kDa PLA2by mass in serum and exudate fluids, e.g. synovial fluid. Herein, we report the utilization of a human recombinant type II 14 kDa PLA2sandwich EIA to directly measure cell or tissue-residing 14 kDa PLA2. It is known that type II 14 kDa PLA2resists acid treatment, and this technique was applied to cell fractions which liberated the enzyme from cellular membrane components prior to quantitation by EIA. Two human immune cell populations were assessed and shown to contain measurable levels of 14 kDa PLA2. Neutrophil or monocyte cytosolic fractions contained no measurable levels whereas the respective 100 000g particulate fractions contained 2.6±0.8 pg (neutrophil) and 2.1±0.6 pg (monocyte) 14 kDa PLA2/μg protein. Human placenta cytosolic fractions contained no measurable levels while 100 000g particulate contained ∼25 ng 14 kDa PLA2/mg protein. This EIA, in conjunction with acid extraction, provides an easy and reproducible assay to identify and quantify this enzyme in cells and whole tissues, expanding our ability to study the relationship of this enzyme to inflammatory processes. |
| Databáze: |
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