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J.H. McDowell, A. Arendt, J.W. Crabb, W.C. Smith; ß–TUBULIN FROM RETINA EXTRACTS BINDS TO ARRESTIN. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3449.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine what retinal proteins bind to arrestin that may be implicated in the light/dark triggered translocation of arrestin in rod photoreceptors. Methods: The synthetic peptide comprising residues 370–404 of bovine arrestin with an additional cysteine added to the amino terminus was synthesized and linked to agarose resin using a SulfoLink kit from Pierce. Another peptide containing the same amino acids, but with a scrambled sequence, was prepared and immobilized on agarose as a control. Bovine retinas were extracted with 0.1 M KPO4, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, pH 7.0 and loaded onto each of the columns. The columns were washed with 0.1 M KPO4, pH 7.0 to baseline. Proteins that bound to the columns were eluted with 0.1 M KPO4, 0.1M NaCl, pH 7.0 and separated by SDS–PAGE. Proteins were excised from the gel, digested in situ with trypsin and identified by capillary LC MS/MS. Arrestin grown with an N–terminal his–tag was immobilized on a 200µl nickel–agarose column. Retinas were extracted with 5 mM Tris–HCl, pH 7.0 and 200 µl extract was loaded onto the arrestin–agarose column. Results: Based on SDS–PAGE Coomassie blue staining, four major proteins eluted from both peptide columns. One protein was more prevalent in the elution from the native column and was identified as ß–tubulin (19 peptides with 42% sequence coverage). One major protein was retarded on the arrestin–nickel column eluting after more than 30 column volumes of wash buffer (10 mM HEPES, 150 mM NaCl, pH 7.0). This protein was also identified as ß–tubulin by western blot analysis. Conclusions: The data show that ß–tubulin binds to arrestin as well as to the C–terminal fragment of arrestin that has previously been shown to be involved in the translocation of arrestin. This implicates the microtubule system in the light/dark induced translocation of arrestin.
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