Properties of membrane-associated sialyltransferase of Escherichia coli.
Properties of membrane-associated sialyltransferase of Escherichia coli.
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大肠杆菌膜相关唾液酸转移酶的特性。
DOI：
10.1016/s0021-9258(19)41995-9
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
F. Troy
中科院分区：
文献类型：
--
作者：
I. K. Vijay;F. Troy
大肠杆菌K-235中的膜相关唾液酸转移酶复合物可以通过脂质缺失解离，并通过添加十一异戊二烯磷酸（一种独特的膜结合脂质辅酶）重新组装。在通过压力崩解和离心分离破坏细胞后，唾液酸转移酶活性与“颗粒”和“可溶性”复合物相关联。动力学研究以及糖核苷酸，金属离子，pH值，硫酸铵和硫醇试剂的要求表明，这两个复合物包含功能相同的酶活性。等密度蔗糖密度梯度离心的研究未分级的总膜上进行的建立，这些唾液酸转移酶的活动与膜的混合物组成的不同的相对量的内部和外部膜。酶定位研究采用DPNH氧化酶，内膜的标记物，和相对磷脂蛋白质组成的测定在两个复合物，提供了额外的支持这一结论。唾液酸聚合物的合成不依赖于其他单糖的掺入，也没有明显的金属离子需求。动力学研究表明，胞苷5-单磷酸-N-乙酰神经氨酸在完整的可溶性和颗粒酶制剂的Km分别为8.1 × 10 ~（-5）M和9.2 × 10 ~（-5）M。类似地，两种酶复合物具有几乎相同的Vmax值。然而，在脱脂酶制剂重新组装后，颗粒酶的Km值增加了10倍，可溶性酶增加了3倍。该增加伴随着Vmax值的大致相同幅度的增加。由于脂质辅酶在完整的酶制剂中是限制性的，因此Vmax的增加反映了重构复合物中活性脂质浓度的增加。外源性添加脂质可以刺激完整膜制剂中唾液酸聚合物的合成。载体脂质的插入取决于温度。在37度时，观察到唾液酸转移酶活性增加120%，而在30%时仅观察到35%的增加。在20度时，未发生刺激。脂相的流动性显然是这种膜相关酶复合物正常发挥功能所必需的。因此，在20度，低于膜脂质转变温度的温度下，脂质相对不动。
Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme. Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex. Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities. Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes. Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion. Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement. Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively. Similarly, both enzyme complexes had nearly identical Vmax values. Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme. This increase was accompanied by an increase of approximately the same magnitude in the Vmax values. Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes. Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid. Insertion of the carrier lipid was dependent on temperature. At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent. At 20 degrees, no stimulation occurred. Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex. Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.