A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex.
Cyclic AMP-dependent protein kinase (PKA) activity was involved in a number of brain functions such as cognitive process or aging. The measurement of PKA activity is traditionally based on the use of [(32)P]ATP in phosphorylation of specific protein. Recently non-isotopic PKA assays have been developed, but none has been tested on brain homogenates. This work aimed to adapt a fluorimetric method of PKA activity into a novel assay never applied before in brain homogenate, and to characterize the enzyme activity and ratio in hippocampus and cortex from rats of different ages. Optimal conditions of homogenization and enzyme protection were determined. The method was sensitive and reproducible (intra-assay and interassay variation was 5.0% and 9.0%, respectively). In hippocampal cytosol, PKA activity was 27+/-8 and 80+/-9 nmol/min per mg protein in basal and cAMP-stimulated activity, respectively, and accounted for 80% of total cell PKA activity. The non-PKA activity, assessed by the use of the PKA specific inhibitor (PKI) accounted for 49.0% and 65.0% of endogenous levels in cytosol and membrane, respectively. cAMP-augmenting drugs effects were measured and increase of 53%, 273% and 118% over basal by 10 microM isoproterenol, 100 microM forskolin, 1 microM Sp-AMP, respectively, was observed. With respect to the changes in animal age, PKA activity increased from newborn to the mature rats but decreased in older rats. The PKA ratio was higher in cytosol than in particulate fraction, and was decreased in hippocampal sample from old rats (P<0.05). This last result was interpreted as related to the loss of cognitive capacities in old animals.
Karege
F
F
Division of Neuropsychiatry, Laboratory of Biochemistry, Geneva University Hospitals, Chemin du Petit Bel-Air 2, CH-1225 Chêne-Bourg (Geneva), Switzerland. karege@sc2a.unige.ch
Schwald
M
M
Lambercy
C
C
Murama
J J
JJ
Cisse
M
M
Malafosse
A
A
D000375:Aging / Q000378:metabolism*; D000818:Animals; D002540:Cerebral Cortex / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006624:Hippocampus / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013379:Substrate Specificity
D009842:Oligopeptides; D012710:Serum Albumin, Bovine; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases