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 approximately 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 degrees C for the artificial substrate, Kemptide, were 38.1+/-4.9 and 3.67+/-0.11 microM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K(m) Mg-ATP values were 61.1+/-6.9 and 30.7+/-4.1 microM. Assay at 4 degrees C lowered the K(m) for Kemptide of E. scudderiana PKAc by 55% and addition of 1M 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 degrees C lowered to just 13-18% of the value at 24 degrees 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 approximately 10 degrees C which suggests a conformational change at low temperature; activation energies (E(a)) were 2.2-3 fold higher for the lower (<10 degrees C) versus higher (>10 degrees C) range. Addition of naturally occurring polyols, 1M glycerol or 0.4M 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.
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Institute of Biochemistry and Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., Canada K1S 5B6.
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D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D020035:Holoenzymes / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*
D020035:Holoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases