title: cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels.
abstract: Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor.
authors.affiliation: Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
authors.forename: J A
authors.initials: JA
authors.lastname: MacDonald
authors.forename: K B
authors.initials: KB
authors.lastname: Storey
mesh_terms: D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D006605:Hibernation / Q000502:physiology*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012589:Sciuridae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D020035:Holoenzymes; D007527:Isoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases; D019791:Guanidine