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Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.

Seyed Tabaei (Institutionen för teknisk fysik, Biologisk fysik) ; Michael Rabe (Institutionen för teknisk fysik, Biologisk fysik) ; Henrik Zetterberg ; Vladimir P. Zhdanov (Institutionen för teknisk fysik, Kemisk fysik) ; Fredrik Höök (Institutionen för teknisk fysik, Biologisk fysik)
Journal of the American Chemical Society (0002-7863). Vol. 135 (2013), 38, p. 14151-8.
[Artikel, refereegranskad vetenskaplig]

Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

Denna post skapades 2013-10-02. Senast ändrad 2015-10-05.
CPL Pubid: 184470


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Institutioner (Chalmers)

Institutionen för teknisk fysik, Biologisk fysik (2007-2015)
Institutionen för neurovetenskap och fysiologi, sektionen för psykiatri och neurokemi (GU)
Institutionen för teknisk fysik, Kemisk fysik (1900-2015)


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