RESUMO
Zn2+ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of 65Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.
Assuntos
Cátions Bivalentes/metabolismo , Proteína Básica da Mielina/metabolismo , Zinco/metabolismo , Animais , Autorradiografia , Bovinos , Colódio , Luz , Microscopia Eletrônica , Ligação Proteica , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
We have previously shown that CNS myelin basic protein (MBP) can be purified in the lipid-bound, native-like form by using a procedure based on myelin solubilization with detergents at pH above 7, and on the filter-like use of hydroxyapatite to separate non-adsorbed MBP from other myelin proteins. Here, we report on the isolation of MBP in the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate (CHAPS), which does not interfere at 280 nm and can be removed by dialysis. This detergent appears to improve MBP purification and to be suitable for fluorescence and reconstitution studies that can be useful to understand both structure and function of MBP in its natural environment.
