Determination of PLDα activity in protein soluble fractions
Protein concentration of the soluble fraction was determined using a
Coomassie Plus Protein Assay (Thermo Scientific). To measure PLDα
activity, 15 µg protein were used per assay. The substrate solution
consisted of 8 µM fluorescent PC (BODIPY-PC, D3792, Life Technologies),
50 µM 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (Avanti Polar
Lipids), 0.015% sodium deoxycholate (MP Biomedicals), and 50 mM MES
buffer (pH 6.5). The substrate solution was shaken gently at 23°C for 30
min, followed by sonication for 10 min. The reaction solution contained
20 mM CaCl2, 0.4% n -butanol (Lachema), and 25 μL
of substrate solution. The reaction was initiated by adding protein
solution, and incubated at 28°C with 300 rpm for 30 min. Lipids were
extracted and run through HP-TLC silica gel-60 plates as in Krčkováet al. (2018). Plates were developed in a mobile phase of
acetone/ethanol (1/1, by vol.) and laser-scanned using a Fuji FLA-7000
fluorescence scanner. Phosphatidylbutanol spots were quantified using
the Fuji FLA-7000 quantification software Multigauge (version (Fujifilm,
Japan). BODIPY-phosphatidylbutanol was identified after comparison to
the standard, prepared using commercially available PLD (Sigma) (Pejcharet al. , 2010).