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.
Funchal
Cláudia
C
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.
de Almeida
Lúcia Maria Vieira
LM
Oliveira Loureiro
Samanta
S
Vivian
Lilian
L
de Lima Pelaez
Priscila
P
Dall Bello Pessutto
Franciele
F
Rosa
Aline Meyer
AM
Wajner
Moacir
M
Pessoa Pureur
Regina
R
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