A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities

Abstract

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

“…Thanks to the presence of β-cyclodextrin, the α-eleostearic acid released from EEPC is solubilized in the aqueous phase. This leads to an increase in the OD at 272 nm that is specific to α-eleostearic acid and allows estimating PLA activity ( El Alaoui et al, 2014 ; El Alaoui et al, 2016 ; Mendoza et al, 2012 ; Serveau-Avesque et al, 2013 ).…”

“…These kinds of differences has been observed for example in the PLA activity determination of rGPLRP2 on C8-PC (1,2-dioctanoyl phosphatidylcholine) and egg PC with values of 551 ± 50 and 812 ± 100 U/mg, respectively, using the pH-stat technique in which the conditions assay consisted of a mechanically stirred emulsion of substrate previously emulsified by ultrasonic treatment ( Dridi et al, 2013 ). In other hand, the PLA activity of rGPLRP2 on coated EOPC (1-α-eleostearoyl-2-octadecyl- rac -glycero-3-phosphocholine) was reported with a value of 0.3 ± 0.02 U/mg ( El Alaoui et al, 2016 ), while the PLA activity of rGPLRP2 with the cHTS-PLA assay developed in this work was of 25.2 ± 0.9, about 84 times more compared with EOPC, and 32 times less compared to pH-stat technique using the same substrate.…”

“…PLA activity assay was based on described methods ( El Alaoui et al, 2014 ; El Alaoui et al, 2016 ; Mendoza et al, 2012 ; Serveau-Avesque et al, 2013 ) with some modifications. The synthetic and specific EEPC substrate ( El Alaoui et al, 2014 ) was first coated in 96-wells UV-microtiter plates using a 0.5 mg/mL solution prepared in ethanol and containing 0.01% BHT as an antioxidant (100 µL/well).…”

A Role of Newly Found Auxiliary Site in Phospholipase A1 from Thai Banded Tiger Wasp (Vespa affinis) in Its Enzymatic Enhancement: In Silico Homology Modeling and Molecular Dynamics Insights

“…Compared with other phospholipase classes, the known PLA1 inhibitor is not commonly reported. The main strategy of PLA1 inhibition is to design a lipid mimetic compound or phospholipid bilayer derivatives [ 21 , 22 ] to compete with a substrate. The idea of the auxiliary site inhibition would be of interest because the site is a solvent-accessible surface.…”

Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization

“…Withers-Martinez et al have modelled a molecule of DGDG in the active site of the known 3D structure of a GPLRP2 chimera and the most favourable steric organization suggested that the ester bond at sn-2 position unlike the ester bond at sn-1 position could not be hydrolyzed by GPLRP2 [58]. More recent studies performed with synthetic acylglycerol substrates bearing non-hydrolysable bonds at sn-1 or sn-2 positions have however shown that rGPLRP2 can hydrolyze the ester bond at sn-2 position at a rate that is 10-fold lower compared to the hydrolysis of the ester bond at sn-1 position [59]. It is not known yet whether GPLRP2 displays a similar regioselective preference on galactolipids, but two hypotheses can be raised concerning the slow hydrolysis of C8-MGMG and the production of MGG observed during the enzymatic hydrolysis of C8-MGDG by rGPLRP2.…”