Byford M F, Bloxham D P
Biochem J. 1984 Oct 15;223(2):359-67. doi: 10.1042/bj2230359.
Incubation of the triazine dye Procion Blue MX-R with L- and M-type pyruvate kinase resulted in rapid time- and dye-concentration-dependent loss of activity. L-type pyruvate kinase was protected only by a low concentration of Mg2+; this was not the case with the M-type enzyme. Modification of the L-type form resulted in the incorporation of 1.54 +/- 0.057 mol of dye/mol of enzyme subunit in the absence of Mg2+, but only 0.73 +/- 0.024 mol of dye/mol of enzyme subunit in the presence of Mg2+. Tryptic peptide mapping of L-type pyruvate kinase modified in the presence and in the absence of Mg2+ further indicated that there were two sites modified in the enzyme, one of which was protected by Mg2+. The pKa of the nucleophile involved in the modification was calculated to be 7.1, implicating the possible involvement of a histidine residue. L-type enzyme was bound to Sepharose-immobilized Procion Blue MX-R specifically in the presence of Mg2+, whereas binding of the M-type enzyme was Mg2+-independent. The specific interaction of L-type pyruvate kinase with the dye was exploited in the large-scale purification of the enzyme and in the isolation of the phosphorylated enzyme.
将三嗪染料普施安蓝MX-R与L型和M型丙酮酸激酶一起温育，会导致酶活性迅速随时间和染料浓度而丧失。L型丙酮酸激酶仅在低浓度Mg2+存在时受到保护；M型酶则不然。在没有Mg2+的情况下，L型酶的修饰导致每摩尔酶亚基掺入1.54±0.057摩尔染料，但在有Mg2+存在时，每摩尔酶亚基仅掺入0.73±0.024摩尔染料。对在有和没有Mg2+存在时修饰的L型丙酮酸激酶进行胰蛋白酶肽图谱分析，进一步表明该酶有两个位点被修饰，其中一个位点受到Mg2+的保护。计算得出参与修饰的亲核试剂的pKa为7.1，这意味着可能有组氨酸残基参与其中。L型酶在Mg2+存在时特异性地结合到固定在琼脂糖上的普施安蓝MX-R上，而M型酶的结合则不依赖Mg2+。L型丙酮酸激酶与染料的特异性相互作用被用于该酶的大规模纯化以及磷酸化酶的分离。
