PARTIAL PURIFICATION AND CHARACTERIZATION OF PROTEIN p83 FROM RAT BRAIN.
Dombroski, T.C.D.1; Ferro, J.A.2; Oliveira, F. S.3;and Martins, A.R. 1
1 Departments of Neurology and Pharmacology, FMRP-USP; 2 Department of Biotecnology, UNESP-Jaboticabal; 3Department of Surgery, FORP-USP
P83 is an apparently novel nervous system specific protein. It has been detected using a polyclonal anti-lectin KM+ antibody, and Western blot in rat and human SNC. Immunoreactive-like p83 was detected in migratory and pre-migratory granule and Purkinjes cells. P83 has an apparent molecular weight 83 kDa, and its expression in the rat cerebellum is development-regulated. P83 interacts with heat shock protein 90 beta, tubulin, Tau and laminin. These properties indicate its biological importance, and also make its purification difficult. A partial characterization of brain p83 is described here. Rat brain (150 g) was homogenized in a hypotonic buffer, and a 24,000g x 2h supernatant was precipitated with 25-50% ammonium sulfate (SA). The SA fraction was purified by column chromatography on DEAE-Sepharose, phenyl- and butyl-Sepharose. The interaction of p83 with ConA-Sepharose column was tested. The assay for p83 consists of a Western blot of a given fraction and its immunodetection using an affinity-purified anti KM+ antibody. The soluble fraction of the rat cerebellum exhibits a single immunoreactive band, whereas the brain soluble fraction exhibits doublets. During purification of brain p83 by DEAE-Sepharose and hydrophobic chromatography, p83 has been detected as a single band, as a doublet and as a triplet by Western blot. These forms of p83 diverged by no more than 3 kDa, and were differentially eluted from both types of columns. Re-chromatography of a pool containing only doublets of p83 on a DEAE-Sepharose column results in separation of single bands and doublets. These data indicate that p83 molecular forms differ by surface charge and hydrophobicity, and suggest that one form could be reversibly converted in another. The Butyl-column eluate, chromatographed in ConA-Sepharose column resulted in imunoreactive form of p83 eluted in the wash of the column. This form was about 55 kDa and will be submitted to SDS-PAGE and colloidal Comassie staining for excision of the band and sequencing.
Supported by FAPESP, FAEPA and CNPQ
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