The use of permeabilized cells to assay protein phosphorylation and catecholamine release

A number of approaches can be used to determine the protein kinases and pro tein phosphatases acting on particular phosphoproteins in vivo. Cell permea bilization represents one such approach. In this overview we discuss the di fferent permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on t he extent of digitonin-permeabilization, protein phosphorylation and catech olamine release are also discussed. The factors include the permeabilizatio n medium, the ions such as calcium, and the second messengers, such as cAMP , IP3, cADPR and calmodulin. The effect of specific peptide inhibitors of p rotein kinases on tyrosine hydroxylase phosphorylation is illustrated. Adva ntages and disadvantages of cell permeabilization procedures are discussed throughout the text.