An assay system for the detection of phospholipase C activity

Abstract

Phospholipase C (PLC, EC 3.1.4.3) enzymes specifically hydrolyze the C-O-P-bond in phospholipids, yielding sn-1,2(2,3)-diglycerides and a phosphate residue bearing the corresponding head group. Biochemical characterization of PLC requires methods for determination of activity. During characterization and purification, proteins are separated by polyacrylamide gel electrophoresis (PAGE). For direct identification and visualization of PLC, a new assay for activity staining in native and renatured SDS-PAGE is described. Incubation of a gel containing an active PLC in the presence of a-naphthylphosphorylcholine leads to a-naphthol formation. This reacts with the diazonium salt Fast Red, forming a red dye which allows clear determination of PLC purity, molecular weight and substrate specificity. The assay was verified using commercially available PC-PLC and new PC-PLC-producing Bacillus cereus strains. The substrate a-NPC was prepared by chemical synthesis at an overall yield of 12%.

Nanoceria-Based Phospholipase-Mimetic Cell Membrane Disruptive Antibiofilm Agents

“…As the nanocatalysts mainly exhibited PLC-like activity, the catalytic parameters were found using model substrate, p -nitrophenyl phosphoryl choline (NPPC) (Figure S7a,b). , The hydrolysis of NPPC using Cnp and PAA-Cnp (100 μg/mL used for both nanocatalysts) was monitored by the UV–vis absorption spectra of the final product p -nitrophenol, which showed absorption maxima at 405 nm.…”

“…As the nanocatalysts mainly exhibited PLC-like activity, the catalytic parameters were found using model substrate, p-nitrophenyl phosphoryl choline (NPPC) (Figure S7a,b). 57,58 The hydrolysis of NPPC using Cnp and PAA-Cnp (100 μg/mL used for both nanocatalysts) was monitored by the UV−vis absorption °C for 36 h. The peaks for POPG appeared at 771.5 (M + Na + ), 793.5 (M + 2Na + ), and 809.4 (M + Na + + K + ) m/z, whereas the peaks at 577 and 701.44 m/z were due to the products formed by the PLC and PLD pathways, respectively. (b) Schematic representation of the UV− vis assay for monitoring the PLC and PLD activity of enzyme using a phosphatidylcholine (PC) substrate.…”

Conjugated Polyelectrolyte Based Real-Time Fluorescence Assay for Phospholipase C

“…A comprehensive literature survey reveals that many assays for monitoring the enzymatic activity of PLC from C. perfringens have been previously reported. ,,,,,,, The previously reported assays are based on a variety of detection methods including turbidimetric, pH-stat titration, radiometric, and continuous fluorometric methods. Concerning the sensitivity, the best detection limit of 0.005 units·mL -1 for PLC from C. perfringens was achieved by using an ELISA assay .…”

“…PLC catalyzes the hydrolysis of the phosphate ester in a phospholipid selectively at the glycerol side, yielding a diacylglycerol (DAG) and a phosphate-containing head group. The ability to quantitatively monitor PLC catalytic activity and inhibition is important as DAGs play a critical role in cell function and the signal transduction cascade in mammalian systems. − Several PLC assays have been developed based on turbidimetric, , pH-stat titration, radiometric, , and continuous fluorometric − methods. However, the turbidimetric and titration assays suffer from low sensitivity, − , and the inherent disadvantage that large quantities of enzyme and substrate are required.…”

“…Fluorometric assays based on the determination of choline 46 or inorganic phosphate 45 offer high sensitivity and are continuous. Although they are advantageous when carrying out studies on enzyme kinetics, most of the fluorometric assays require synthetic fluorogenic substrates, − ,− which results in low catalytic turnover of enzymes and reduced reaction rates. , …”