Immobilization of the restriction endonuclease PstI

PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity. It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible for inactive enzyme preparations.     

9个PstI接头片段的合成及其酶反应研究

采用固相亚磷酸三酯法和化学与酶促相结合的方法合成了９个ＰｓｔＩ接头片段，并对它们进行了酶切反应研究．结果表明：ＰｓｔＩ识别序列中尿苷的存在只降低其互补链的切割程度，对其本链没有影响；ＰｓｔＩ切割底物是按两步单股切割机制进行，作用的最小底物是含有识别序列的８～１２聚脱氧核糖核苷酸，且对底物中识别序列两边的碱基对数目有一定要求；ＰｓｔＩ识别序列中的二个胞苷在酶反应中起的作用是不一样的