Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects

Abstract

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 milligram protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. K m values at 24°C for the artificial substrate, Kemptide, were 38.1±4.9 and 3.67±0.11 μM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K m Mg-ATP values were 61.1±6.9 and 30.7±4.1 μM. Assay at 4°C lowered the K m for Kemptide of E. scudderiana PKAc by 55% and addition of 1 M glycerol further lowered the K m. Low assay temperature also enhanced holoenzyme dissociation in both species with the K a value for cyclic 3′5′-monophosphate at 4°C lowered to just 13–18% of the value at 24°C. 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 ( E a) were 2.2–3 fold higher for the lower (<10°C) versus higher (>10°C) range. Addition of naturally occurring polyols, 1 M glycerol or 0.4 M sorbitol, affected E a 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.