Ortiz A I, Reglero A, Rodríguez-Aparicio L B, Luengo J M
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Eur J Biochem. 1989 Jan 2;178(3):741-9. doi: 10.1111/j.1432-1033.1989.tb14505.x.
The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.
对从在化学限定培养基（适合产大肠杆菌K抗原多糖酸）中生长的大肠杆菌K - 235获得的膜结合唾液酸转移酶进行了研究。计算得出该酶在体内的半衰期为20小时。动力学测试表明（在33℃和pH 8.3条件下），该酶对CMP - Neu5Ac呈现双曲线行为（Km为250μM），转变温度为31.3℃。该酶受到NH4 +、一些二价阳离子以及几种与巯基反应的试剂的抑制。去污剂和脂肪酸也会抑制唾液酸转移酶的活性。通过阻止[14C]Neu5Ac掺入蛋白质 - 复合物中间体进而掺入游离聚合物中，CMP强烈抑制了大肠杆菌K抗原多糖酸的体外合成。CDP和CTP也抑制（分别为91%和84%）该酶的活性，而胞嘧啶和胞苷则无影响。CMP抑制符合竞争模型，计算得出的Ki为30μM。蛋白质[14C]Neu5Ac与CMP、CDP和CTP一起孵育导致了CMP - [14C]Neu5Ac的从头合成。通常会使反应平衡向聚合物合成方向移动的大肠杆菌K抗原多糖酸的存在，并不影响这种CMP - [14C]Neu5Ac的从头形成。CMP在体内也抑制大肠杆菌K抗原多糖酸的生物合成。
