Rodríguez-Aparicio L B, Reglero A, Luengo J M
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Biochem J. 1987 Sep 1;246(2):287-94. doi: 10.1042/bj2460287.
Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.
在37℃下，于pH 7.0的0.1 M - Tris/马来酸缓冲液中，对大肠杆菌K - 235摄取N - 乙酰[4,5,6,7,8,9 - ¹⁴C]神经氨酸进行了体内动力学测量。在这些条件下，摄取至少30分钟呈线性，计算得出的唾液酸Km值为30微摩尔。该转运系统对渗透休克敏感，并且受到氧化磷酸化解偶联剂[2,4 - 二硝基苯酚（100%）；叠氮化钠（66%）]以及代谢抑制剂氰化钾（84%）和砷酸钠（76%）的强烈抑制。含硫醇的化合物巯基乙醇、谷胱甘肽、半胱氨酸、二硫苏糖醇和胱氨酸对唾液酸转运速率没有显著影响，而硫醇修饰试剂N - 乙基马来酰亚胺、碘乙酸和对氯汞苯甲酸几乎完全阻断（大于94%）了这种N - 乙酰糖的摄取。N - 乙酰葡糖胺非竞争性抑制N - 乙酰神经氨酸的转运，而其他碳水化合物（己糖、戊糖、己糖醇、己糖醛酸、二糖、三糖）以及N - 乙酰糖或氨基酸衍生物（N - 乙酰甘露糖胺、N - 乙酰半胱氨酸、N - 乙酰脯氨酸和N - 乙酰谷氨酸）没有任何影响。令人惊讶的是，L - 甲硫氨酸及其非硫类似物L - 正亮氨酸部分阻断了这种糖的转运（50%），而D - 甲硫氨酸、D - 正亮氨酸、几种L - 甲硫氨酸衍生物（L - 甲硫氨酸甲酯、L - 甲硫氨酸乙酯、L - 甲硫氨酸亚砜）和其他氨基酸不影响唾液酸的摄取。N - 乙酰神经氨酸转运系统由唾液酸诱导，并且受到用于大肠杆菌生长的碳源严格调控，阿拉伯糖、乳糖、葡萄糖、果糖和葡糖胺是对该系统抑制作用最大的碳水化合物。向培养液中添加环磷酸腺苷可逆转葡萄糖的作用，表明N - 乙酰神经氨酸摄取系统处于分解代谢调控之下。唾液酸转运不需要蛋白质合成。
