CAMP-DEPENDENT PROTEIN-KINASE FROM BROWN ADIPOSE-TISSUE - TEMPERATUREEFFECTS ON KINETIC-PROPERTIES AND ENZYME ROLE IN HIBERNATING GROUND-SQUIRRELS

Arousal from hibernation requires thermogenesis in brown adipose tissu e, a process that is stimulated by P-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 respir ation. To determine whether specific adaptations (perhaps temperature- dependent) facilitate PKA kinetic properties or protein-phosphorylatin g ability, the catalytic subunit of PKA (PKAc) from interscapular brow n adipose of the ground squirrel Spermophilus richardsonii, was purifi ed (final specific activity = 279 nmol phosphate transferred per min p er 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 numer ous significant effects on ground squirrel PKAc including: (a) pH opti mum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K-m value s at 37 degrees C for Mg.ATP (49.2 +/- 3.4 mu M) and for two phosphate accepters, Kemptide (50.0 +/- 5.5 mu M) and Histone IIA (0.41 +/- 0.0 5 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temper ature change had little or no effect on K-m values of rabbit PKAc, sug gesting a specific positive thermal modulation of the hibernator enzym e. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and a ctivation energies that were 29.1 +/- 1.0 kJ/mol for temperatures >9 d egrees 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 degre es C with a 13-fold higher activation energy over the lower temperatur e range. However, fluorescence analysis of PKAc in the absence of subs trates, showed a linear change in fluorescence intensity and wavelengt h of maximal fluorescence over the entire temperature range; this sugg ested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also aff ected the Hill coefficient for cAMP dissociation of the ground squirre l 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 grou nd squirrel brown adipose with P-32-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the pat terns of phosphoproteins, both between euthermic and hibernating anima ls as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylatio n 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 groun d squirrel PKAc kinetic properties, suggest differential control of th e enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enz yme during arousal from torpor.