Development of an assay for phospholipase C using column-reconstituted, extruded phospholipid vesicles.
The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta activity. This approach involves reconstitution of purified betagamma dimers into extruded phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed betagamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of betagamma dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the gamma subunits in the activation of PLC-beta. The beta1gamma1 dimer (containing the farnesyl group) and the beta1gamma2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The beta1gamma2 dimer is more potent and effective in stimulating PLC-beta activity than the beta1gamma1 dimer. The EC50 values of betagamma dimers for the activation of PLC-beta determined with this method were lower than those determined by previous methodology, showing that betagamma subunits have a subnanomolar affinity for PLC-beta.
Myung
C S
CS
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. cm8r@virginia.edu
Paterson
A
A
Harden
T K
TK
Garrison
J C
JC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003902:Detergents; D019281:Dimerization; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000032:analysis*; D008081:Liposomes; D008854:Microscopy, Electron; D054799:Phospholipase C beta; D010743:Phospholipids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera; D010738:Type C Phospholipases / Q000032:analysis*
D003902:Detergents; D007527:Isoenzymes; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins