Rapid and serial determination of protein kinase C activity and of the associated [3H]PDBu binding using a 96-well microtiter plate and a cell harvester.
We propose a serial assay of both protein kinase C activity and the related [3H]phorbol 12,13-dibutyrate binding, each carried out in 96-multiwell dishes, started and stopped row by row using a multipipet. Protein kinase C activity is observed through the transfer of the gamma-phosphoryl group of radioactive ATP onto histone H1 type III-S. Enzymatic reactions are started by adding enzyme extracts and stopped by adding trichloroacetic acid. Acidic precipitates of each row are simultaneously collected on glass fiber paper using a cell harvester. The addition of bovine serum albumin and cold ATP at the end of the reaction and the addition of trichloroacetic acid in the washing fluid lead to a high recovery of protein kinase C activity and reproducible results. Measurement of [3H]phorbol 12,13-dibutyrate binding to protein kinase C was carried out in a mixed micellar solution as described elsewhere (Y. Hannun and R. M. Bell (1987) in Methods in Enzymology, Vol. 141, pp. 287-293). The quaternary complex formed from protein kinase C, phosphatidylserine, calcium, and [3H]phorbol 12,13-dibutyrate was then bound to a beaded anionic exchanger which was automatically separated from the free phorbol 12,13-dibutyrate by microfiltration using a cell harvester. The binding reaction was highly calcium- and phosphatidylserine-dependent and calcium had to be added to washing fluid for optimal recovery. Determination of protein kinase C activity and phorbol 12,13-dibutyrate binding gave results similar to those of other published methods and the signal/noise ratio was greatly increased. Using a semi-automated cell harvester, the system is partially automated and provides accurate and reproducible results.
Parant
M R
MR
CNRS U.A. 530, INSERM U. 58, Montpellier, France.
Vial
H J
HJ
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D011232:Chemical Precipitation; D004789:Enzyme Activation; D006168:Guinea Pigs; D008832:Microchemistry / Q000295:instrumentation*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000032:analysis*
D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D002118:Calcium