Protein kinase A: Purification and characterization of the enzyme from two cold-hardy goldenrod gall insects
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. 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.
|Keywords||Epiblema scudderiana, Eurosta solidaginis, Freeze avoidance, Freeze tolerance, Glycogen metabolism, Polyol synthesis|
|Journal||Insect Biochemistry and Molecular Biology|
Pfister, T.D., & Storey, K. (2002). Protein kinase A: Purification and characterization of the enzyme from two cold-hardy goldenrod gall insects. Insect Biochemistry and Molecular Biology, 32(5), 505–515. doi:10.1016/S0965-1748(01)00128-X