4] Measurement of hormone-stimulated protein phosphorylation in intact cells

Abstract

This chapter discusses the measurement of hormone-stimulated protein phosphorylation in intact cells. The use of radioactive phosphate to measure the phosphorylation state of enzymes in intact cells began in 1956, using the approach to demonstrate that epinephrine and glucagon stimulated the phosphorylation of phosphorylase in dog liver slices. The recent development of sodium dodecyl sulfate (SDS)-polyacrylamide gel systems capable of resolving very large numbers of proteins has increased the utility of this technique and it is now being applied to a number of tissues responsive to hormones or neurotransmitters. The advantages of measuring changes in protein phosphorylation in intact cells are (1) the protein kinases and substrates are in their native state during the phosphorylation reaction, obviating arguments about the relevance of the phosphorylation events and (2) changes can be observed in the phosphorylation states of proteins when the hormone message or the protein kinase mediating the reaction are not clearly defined.