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AIM To investigate the interaction of Zot withmicrotubule.METHODS Zot affinity column was applied topurify Zot-binding protein(s)from crudeintestinal cell lysates.After incubation at roomtemperature,the column was washed and theproteins bound to the Zot affinity column wereeluted by step gradient with NaCl(0.3 mol·L~(-1)-0.5mol·L~(-1)).The fractions were subjected to6.0%-15.0%(w/v)gradient SDS-PAGE andthen transferred to PVDF membrane for N-terminal sequencing.Purified Zot and tauprotein were blotted by using anti-Zot or anti-tauantibodies.Finally,purified Zot was tested in anin vitro tubulin binding assay.RESULTS Fractions from Zot affinity columnyielded two protein bands with a Mr of 60 kU and45kU respectively.The N-terminal sequence ofthe 60 kU band resulted identical to β-tubulin.Zot also cross-reacts with anti-tau antibodies.Inthe in vitro tubulin binding assay,Zot co-precipitate with Mt,further suggesting that Zotpossesses tubulin-binding properties.CONCLUSION Taken together,these resultssuggest that Zot regulates the permeability ofintestinal tight junctions by binding tointracellular Mt,with the subsequent activationof the intracellular signaling leading to thepermeabilization of intercellular tight junctions.
AIM To investigate the interaction of Zot with microtubule. METHODS Zot affinity column was applied topurify Zot-binding protein (s) from crudeintestinal cell lysates. After incubation at room temperature, the column was washed and the proteins bound to the Zot affinity column wereeluted by step gradient with NaCl (0.3 mol·L -1 - 0.5 mol·L -1). The fractions were subjected to 6.0% -15.0% (w / v) gradient SDS-PAGE and transferred to a PVDF membrane for N -terminal sequencing. Purified Zot and tauprotein were blotted by using anti-Zot or anti-tau antibodies. Finaally, purified Zot was tested in an in vitro tubulin binding assay. RESULTS Fractions from Zot affinity columnyielded two protein bands with a Mr of 60 kU and 45 kU respectively The N-terminal sequence of the 60 kU band due identical to β-tubulin. Also to cross-reacts with anti-tau antibodies. Inthe in vitro tubulin binding assay, Zot co-precipitate with Mt, further suggesting that Zotpossesses tubulin-binding properties .CONCLUSION Taken together, t hese resultssuggest that Zot regulates the permeability of intestinal tight junctions by binding to intracellular junctions, with the next activation of the intracellular signaling leading to the permeabilization of intercellular tight junctions.