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Molecular Interaction of S‐100 Proteins with Microtubule Proteins In Vitro
Author(s) -
Donatq Rosario,
Giambanco Ileana,
Aisa Maria Cristina
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb07371.x
Subject(s) - tubulin , nitrocellulose , biotinylation , microtubule , chemistry , gel electrophoresis , biochemistry , microtubule associated protein , colchicine , in vitro , polyacrylamide gel electrophoresis , trypsin , microbiology and biotechnology , biology , enzyme , membrane , genetics
Several procedures were employed to examine the in vitro interaction between S‐100 proteins and microtubule proteins. Binding of S‐100 to τ factors was observed under all experimental conditions. S‐100 binding to microtubule‐associated protein 2 (MAP2) was best detected by exposing nitrocellulose‐immobilized MAP2 or MAPs to either 125 I‐labeled S‐100 or biotinylated S‐100. S‐100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the Afunctional cross‐linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate‐polyacrylamide gel [electrophoresis (SDS‐PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S‐100 and tubulin, as well as between S‐100 and a relatively low‐molecular‐weight MAP, was evidenced by immunoblotting using an anti‐S‐100 antiserum. Alternatively, complex formation between biotinylated S‐100 and either tubulin or MAPs was visualized by means of avidin‐peroxidase, after SDS‐PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S‐100 and tubulin was strictly Ca 2+ dependent, and resistant to high concentrations of KC1, colchicine, or vinblastine.