Endogenous phosphorylation in vitro

Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins

Richard G. Conway, Aryeh Routtenberg*

*Corresponding author for this work

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [ 32 P]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, F 1 and F 2 , 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 F 1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F 2 was relatively stable. While phosphorylation of F 2 was also stable 24 h post-mortem, Band F 1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F 2 somewhat, it eliminated the in vitro phosphorylation of Band F 1 . 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), F 1 (M R 47,000-49,000) and F 2 (M R 40,000-45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F 1 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 F 1 , however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F 1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F 1 is: (3) increased in phosphorylation in liquid nitrogen P 2 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 F 1 phosphorylation may be Ca 2+ -dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F 1 in neuronal function.

Original languageEnglish (US)
Pages (from-to)313-324
Number of pages12
JournalBrain Research
Volume170
Issue number2
DOIs
StatePublished - Jul 13 1979

Fingerprint

Anesthesia
Phosphorylation
Brain
Proteins
Synaptosomes
Pentobarbital
In Vitro Techniques
Nitrogen
Gels
Righting Reflex
Decapitation
Densitometry
Phosphoproteins
Osmotic Pressure
Immersion
Coma
Cerebral Cortex
Cyclic AMP
Electrophoresis
Phosphates

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

Cite this

@article{c26f2a580fd2406887fee8c8d6d347e1,
title = "Endogenous phosphorylation in vitro: Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins",
abstract = "In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [ 32 P]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, F 1 and F 2 , 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 F 1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F 2 was relatively stable. While phosphorylation of F 2 was also stable 24 h post-mortem, Band F 1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F 2 somewhat, it eliminated the in vitro phosphorylation of Band F 1 . 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), F 1 (M R 47,000-49,000) and F 2 (M R 40,000-45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F 1 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 F 1 , however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F 1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F 1 is: (3) increased in phosphorylation in liquid nitrogen P 2 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 F 1 phosphorylation may be Ca 2+ -dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F 1 in neuronal function.",
author = "Conway, {Richard G.} and Aryeh Routtenberg",
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Endogenous phosphorylation in vitro : Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins. / Conway, Richard G.; Routtenberg, Aryeh.

In: Brain Research, Vol. 170, No. 2, 13.07.1979, p. 313-324.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Endogenous phosphorylation in vitro

T2 - Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins

AU - Conway, Richard G.

AU - Routtenberg, Aryeh

PY - 1979/7/13

Y1 - 1979/7/13

N2 - In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [ 32 P]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, F 1 and F 2 , 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 F 1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F 2 was relatively stable. While phosphorylation of F 2 was also stable 24 h post-mortem, Band F 1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F 2 somewhat, it eliminated the in vitro phosphorylation of Band F 1 . 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), F 1 (M R 47,000-49,000) and F 2 (M R 40,000-45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F 1 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 F 1 , however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F 1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F 1 is: (3) increased in phosphorylation in liquid nitrogen P 2 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 F 1 phosphorylation may be Ca 2+ -dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F 1 in neuronal function.

AB - In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [ 32 P]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, F 1 and F 2 , 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 F 1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F 2 was relatively stable. While phosphorylation of F 2 was also stable 24 h post-mortem, Band F 1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F 2 somewhat, it eliminated the in vitro phosphorylation of Band F 1 . 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), F 1 (M R 47,000-49,000) and F 2 (M R 40,000-45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F 1 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 F 1 , however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F 1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F 1 is: (3) increased in phosphorylation in liquid nitrogen P 2 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 F 1 phosphorylation may be Ca 2+ -dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F 1 in neuronal function.

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