Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles.
Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles.
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DOI：
10.1016/s0021-9258(17)44521-2
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发表时间：
1983-09
期刊：
影响因子：
--
通讯作者：
J. E. Leonard;M. Saier
中科院分区：
文献类型：
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作者：
J. E. Leonard;M. Saier
纯化的甘露醇酶II从大肠杆菌中重建磷脂囊泡采用辛基葡萄糖苷稀释程序，并显示催化载体甘露醇1-磷酸：甘露醇转磷酸。脂质体中的酶的重建表现出显着的依赖于辛基葡糖苷浓度，最佳浓度为1.2%。重建的转磷酸活性表现出绝对依赖甘露醇1-磷酸作为磷酰基供体，是敏感的N-乙基马来酰亚胺，并有一个接近6的最佳pH值。通过加入50 μ M未标记甘露醇或0.5%十二烷基硫酸钠，可从脂蛋白体中释放囊内放射性标记的甘露醇磷酸盐。作为甘露醇酶II浓度的函数测量的脂质体内甘露醇磷酸盐的形成速率显示S形响应，表明在高酶浓度下甘露醇酶II以聚集或低聚状态存在，并且这种形式在催化载体甘露醇转磷酸化反应中比酶的单体或解离形式更有活性。
Purified mannitol Enzyme II from Escherichia coli was reconstituted in phospholipid vesicles employing the octylglucoside dilution procedure and was shown to catalyze vectorial mannitol 1-phosphate:mannitol transphosphorylation. Reconstitution of the enzyme into liposomes showed a marked dependency upon the octylglucoside concentration with an optimum at 1.2%. The reconstituted transphosphorylation activity exhibited an absolute dependence upon mannitol 1-phosphate as the phosphoryl donor, was sensitive to N-ethylmaleimide, and had a pH optimum near 6. The intravesicular radiolabeled mannitol phosphate could be released from the proteoliposomes by the addition of either 50 microM unlabeled mannitol or 0.5% sodium dodecyl sulfate. The rate of formation of intraliposomal mannitol phosphate, measured as a function of the mannitol Enzyme II concentration, showed a sigmoidal response, suggesting that at high enzyme concentrations the mannitol Enzyme II exists in an aggregated or oligomeric state and that this form is more active than the monomeric or dissociated form of the enzyme in catalyzing the vectorial mannitol transphosphorylation reaction.