21] Purification of Drosophila cAMP-dependent protein kinase

Abstract

In crude extracts of Drosophila adults it was observed that the regulatory subunit of cAMP-dependent protein kinase was rapidly degraded to a 40,000-D form. This proteolysis also occurred in extracts of Drosophila heads, thoraxes, abdomens, and legs. Although proteolytic degradation could be retarded by the simultaneous addition of several protease inhibitots, it still precluded purification of the intact enzyme. Thus, the purification scheme that was devised reflected the necessity to both reduce proteolytic activity and employ procedures that could be accomplished rapidly. The yield of homogeneous Drosophila cAMP-dependent protein kinase is about 100-fold less than from beef heart. Fresh extracts from Drosophila contain the same specific activity of cAMP-dependent histone kinase as beef heart extracts but because the specific activity of the homogeneous fly kinase is three times higher than the cow kinase, there is substantially less enzyme in Drosophila extracts. This higher specific activity is not surprising for a poikilotherm that grows optimally at 25°, because the assays were conducted at 30°. The major difficulty, however, in purifying the Drosophila enzyme arises from proteolytic degradation of the regulatory subunit that occurs throughout the purification.