25] Radiolabeling and detection methods for studying metabolism of regulatory subunit of cAMP-dependent protein kinase I in intact cultured cells

Abstract

Publisher Summary This chapter discusses procedures for combining metabolic labeling with two-dimensional gel analysis to study the regulation of R 1 metabolism. Alterations in expression of cAMP-dependent protein kinase subunits accompanying kinase activation, tissue differentiation, cellular transformation, cell cycle traverse, or somatic mutations have generated interest in kinase subunit metabolism and its intracellular regulation. The most direct approach for studying specific protein metabolism in intact cells involves incorporation of radiolabeled amino acids and subsequent quantitation of radioactivity associated with the protein of interest. Protein modifications can be followed using radioisotopes specific for the modifying groups or, for some modifications, by alterations in electrophoretic mobility of the protein. There has been considerable success using high-resolution two-dimensional polyacrylamide gel electrophoresis of crude cell extracts to resolve type I regulatory subunit (R 1 ) of protein kinase for studies of its synthesis and degradation in cultured S49 mouse lymphoma cells.