Phosphorothiolate analogues of phosphatidylinositols as assay substrates for phospholipase C.
Accurate measurement of phosphatidylinositol-specific phospholipase C (PI-PLC) activity is important in view of the key role of this enzyme in signal-transduction pathways. In this work we synthesized enantiomerically pure phosphorothiolate analogues of all natural PI-PLC substrates, including those of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), 4-phosphate (PI-4-P), 5-phosphate (PI-5-P) and unphosphorylated PI, in both long- and short-chain versions. The enzymatic cleavage of these substrates produces thiol analogues of diacyl glycerol, which can be quantified by UV absorbance after treatment with dipyridyl disulfide. The monodisperse dihexanoyl derivatives are suitable substrates for PI-PLC assay: they give rise to high enzyme activity, and provide excellent linear kinetic responses. For all substrates, we found a good linear correlation between the reaction rate and the amount of enzyme; this indicated the suitability of this assay for enzyme quantification. The short-chain substrates enable the enzyme specificity with variously phosphorylated inositol head groups to be established--unobstructed by substrate aggregation, "scooting" kinetics on micelles, or surface dilution effects. The kinetic results indicated allosteric behavior of PLC for all substrates tested. We found that substrates phosphorylated at the inositol 4-position (phosphorothiolate analogues of PI-4,5-P2 and PI-4-P) displayed very similar kinetic properties, and were cleaved with approximately 20- to 30-fold higher activity than the 4-nonphosphorylated substrates (analogues of PI-5-P and PI). Hence it appears that interactions between the enzyme and the 4-phosphate group of the substrate, but not its 5-phosphate group, is important for PI-PLC catalysis. In addition, the binding affinities of all four substrate types were found to be quite similar; this indicates that the energy of enzyme interaction with the 4-phosphate group is directed almost entirely to catalysis.
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago IL 60612, USA.
Yinghui
Y
Liu
Cornelia
C
Mihai
Robert J
RJ
Kubiak
Mario
M
Rebecchi
Karol S
KS
Bruzik
D002384:Catalysis; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D013379:Substrate Specificity; D010738:Type C Phospholipases
D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C