Pfister, T.D. and Storey, K.B. 2002. Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects. Insect Biochem. Mol. Biol. 32, 505-515.

Protein Kinase A: Purification and Characterization of the Enzyme from Two Cold-hardy Goldenrod Gall Insects.

Thomas D. Pfister and Kenneth B. Storey


The catalytic subunit of protein kinase A (PKAc) was purified to apparent homogeneity from two species of cold-hardy goldenrod gall insects, Epiblema scudderiana and Eurosta solidaginis. Final specific activity for both enzymes was ~74.5 nmol of phosphate transferred per minute per mg protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. Km values at 24C for the artificial substrate, Kemptide, were 38.1 4.9 and 3.67 0.11 M for E. scudderiana and E. solidaginis PKAc, respectively, whereas Km Mg-ATP values were 61.1 6.9 and 30.7 4.1 M. Assay at 4C lowered the Km for Kemptide of E. scudderiana PKAc by 55% and addition of 1 M glycerol further lowered the Km. Low assay temperature also enhanced holoenzyme dissociation in both species with the Ka value for cyclic 3'5'-monophosphate at 4C lowered to just 13-18% of the value at 24C. Low temperature did not affect affinity for Mg-ATP or inhibition by PKA inhibitors (PKAi, H7, H89) but increased inhibition by some salts. PKAc from both species showed a break in the Arrhenius relationship at ~10 C which suggests a conformational change at low temperature; activation energies (Ea) were 2.2-3 fold higher for the lower (<10C) versus higher (>10C) range. Addition of naturally occurring polyols, 1 M glycerol or 0.4 M sorbitol, affected Ea in some cases. Temperature dependent regulation of holoenzyme dissociation and PKAc kinetic properties may have an role in regulating the enzymes involved in polyol synthesis in cold-hardy insects.