Development of a high-throughput assay for two inositol-specific phospholipase Cs using a scintillation proximity format.
Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.
Bembenek
Michael E
ME
Mellennium Pharmaceuticals, Inc, Cambridge, MA 02139, USA. bembenek@mpi.com
Jain
Sadhana
S
Prack
Andrea
A
Li
Ping
P
Chee
Linda
L
Cao
Wei
W
Spurling
Heidi
H
Roy
Rebecca
R
Fish
Susan
S
Rokas
Mihail
M
Parsons
Thomas
T
Meyers
Rachel
R
D000818:Animals; D002417:Cattle; D006801:Humans; D007294:Inositol / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D010716:Phosphatidylinositols / Q000737:chemistry; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods; D013552:Swine; D013997:Time Factors; D014316:Tritium; D010738:Type C Phospholipases / Q000737:chemistry*
D007527:Isoenzymes; D010716:Phosphatidylinositols; D014316:Tritium; D007294:Inositol; D010738:Type C Phospholipases