RAPID AND SERIAL DETERMINATION OF PROTEIN KINASE-C ACTIVITY AND OF THE ASSOCIATED [H-3] 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 γ-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. © 1990.