Thomas D. Pfister and Kenneth B. Storey
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 mg protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. Km 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 Km Mg-ATP values were 61.1 ± 6.9 and 30.7 ± 4.1 µM. Assay at 4°C 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 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 (Ea) 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 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.