In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats.
Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [gamma(32)P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, 90035-003 Porto Alegre, RS, Brazil.
Cláudia
C
Funchal
Lúcia Maria Vieira
LM
de Almeida
Samanta
S
Oliveira Loureiro
Lilian
L
Vivian
Priscila
P
de Lima Pelaez
Franciele
F
Dall Bello Pessutto
Aline Meyer
AM
Rosa
Moacir
M
Wajner
Regina
R
Pessoa Pureur
D000255:Adenosine Triphosphate; D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry / Q000302:isolation & purification; D006651:Histocytochemistry / Q000295:instrumentation / Q000379:methods*; D008297:Male; D016900:Neurofilament Proteins / Q000737:chemistry / Q000302:isolation & purification; D009924:Organ Culture Techniques / Q000379:methods*; D010710:Phosphates; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010759:Phosphorus Isotopes; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000737:chemistry*; D014746:Vimentin / Q000737:chemistry / Q000302:isolation & purification
D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D016900:Neurofilament Proteins; D010710:Phosphates; D010759:Phosphorus Isotopes; D014746:Vimentin; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D010744:Phosphoric Monoester Hydrolases