Endogenous phosphorylation in vitro: Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins

In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [³²P]Labelled phosphoproteins were separated by SDS-slab gel electrophoresis, and the autoradiographs were analyzed by densitometry. We report here that Band F of our previous reports can be separated into two components, F₁ and F₂, using an improved gel system. This separation is particularly relevant in this report since these components appear to be differentially sensitive to the manipulations used. Specifically, we found that while F₁ phosphorylation was markedly diminished by deep barbiturate anesthesia, F₂ was relatively stable. While phosphorylation of F₂ was also stable 24 h post-mortem, Band F₁ phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F₂ somewhat, it eliminated the in vitro phosphorylation of Band F₁. We found that under light barbiturate anesthesia, just at the time when the animals lost the righting reflex, the in vitro phosphorylation of Bands D (M_R 78,000-80,000 daltons), F₁ (M_R 47,000-49,000) and F₂ (M_R 40,000-45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F₁ was depressed in tissue prepared from animals that were deeply comatose. These effects of pentobarbital were more pronounced when animals were sacrificed by liquid nitrogen immersion, rather than by decapitation. Cyclic AMP-dependent phosphorylation of Band D exhibited remarkable stability 24 h post-mortem (7 days in one case), even when brain tissue was left at room temperature (21-23 °C). Phosphorylation of Band F₁, however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F₁ is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F₁ is: (3) increased in phosphorylation in liquid nitrogen P₂ preparations, and may be (4) cAMP-independent, (5) rapidly turning over its phosphate in vivo, and (6) altered by a training experience. Other evidence suggests that: (7) Band F₁ phosphorylation may be Ca²⁺-dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F₁ in neuronal function.