Assay to measure PLCz1 activity

[EveCarter]

A key requirement for finding an inhibitor of PLCz1 is to have an assay that will measure the activity of the protein against different compounds.

Previously used PLC activity assays use tritium labelled phosphatidylinositol 4,5-biphosphate (PIP2), as the protein is known to hydrolyse PIP2 into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). The results are quantified using liquid scintillation spectrofluorimetry. J. Biol. Chem. 2005, 280(35), 31011–31018 doi:10.1074/jbc.M500629200 Biochem. J. 2017, 474, 1003–1016 doi:10.1042/BCJ20161057

There are some commercially available colorimetric assays that provide a chromogenic compound and lyophilised PLC which can be mixed with a tissue sample and provide colorimetric measurement of activity at 405 nm. https://www.abcam.com/phospholipase-c-activity-assay-kit-colorimetric-ab273343.html

We would rather not work with radioactive compounds and, due to the large number of activity tests we plan on doing, the commercial option is probably too expensive, therefore an alternative assay would be desired.

[mattodd]

Interesting. I think we need help here from some of the assay folks elsewhere in the group, as well as Karl Swann's group in Cardiff. Let's see if we can get some of them on here to advise. Agree that fluorometric WAY preferable to radiolabelled.

[EveCarter]

In discussion with Karl Swann yesterday, he suggested looking at the Sondek group's research. They have two fluorogenic reporters that seem to be general for all PLC isoforms: WH-15 and XY-69. WH-15 is soluble and does not selectively partition into phospholipid membranes like PIP2, whereas XY-69 preferentially partitions into membranes therefore can be used to measure the activity of PLCs as either purified enzymes or in cellular lysates.

• J. Biol. Chem. 2013, 288 (8), 5840–5848 doi:10.1074/jbc.M112.422501
  • J. Biol. Chem. 2018, 293 (5), 1728–1735 doi:10.1074/jbc.RA117.000926
  • Phosphoinositides; 2021; Vol. 2251, pp 225–236 doi:10.1007/978-1-0716-1142-5
  • XY-69 is commercially available from Avanti Polar Lipids

There is some more detailed information on the wiki page.

[EveCarter]

A discussion with Matilda Katan has highlighted another assay to measure PLC activity. It uses commercially available Aldol 518 as a substrate for hydrolysis and Aldol 355 as a fluorescence enhancer. Hydrolysis was measured in a continuous format with excitation at 510 nm and emission at 618 nm.

• Sci. Adv. 2022, 8 (25), 1-16 doi:10.1126/sciadv.abp9688

[EveCarter]

Closing as this issue has been resolved. The relevant information and a link to this issue has been included on the wiki here.