cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels |
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Authors: | J A MacDonald K B Storey |
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Institution: | (1) Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6 e-mail: kbstorey@ccs.carleton.ca; Tel.: +1-613-520-3678; Fax: +1-613-520-4497, CA |
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Abstract: | Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-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 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground
squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) Km values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml)
decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on Km 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 °C and activation energies that
were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the
rabbit enzyme showed a breakpoint at 17 °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 °C to 2.19 ± 0.07 at 5 °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 °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.
Accepted: 10 July 1998 |
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Keywords: | Mammalian hibernation PKA catalytic subunit Spermophilus richardsonii Brown adipose thermogenesis |
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