Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.
Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.
复制标题
使用原核衍生探针鉴定新生儿神经元膜中的聚唾液酸。
DOI：
10.1073/pnas.81.7.1971
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发表时间：
1984
影响因子：
11.1
通讯作者：
Troy,FA
中科院分区：
文献类型：
--
作者：
Vimr,ER;McCoy,RD;Vollger,HF;Wilkison,NC;Troy,FA
已经开发了三种原核衍生的探针来鉴定和研究神经元组织中多聚唾液酸单位的时间表达。多克隆抗体、噬菌体衍生的内神经氨酸酶和大肠杆菌K1唾液酸转移酶都对识别或合成含有α-2，8-酮糖苷键的聚（唾液酸）具有特异性。聚唾液酸的免疫反应性与明显的MR值为180，000 - 240，000是特定的发展中的神经元组织，它没有检测到在新生儿肝脏或肾脏或成人脑组织。聚（唾液酸）的发育调节消失与此处描述的识别神经元细胞粘附分子（N-CAM）的聚唾液酸糖基碳水化合物单元的探针一致。用α-2，8-连接的聚唾液酸单位特异性的噬菌体衍生的内神经氨酸酶处理脑提取物消除了免疫反应性。分离由内神经氨酸酶溶解的材料，用硼三肽还原，并显示含有具有3至6个唾液酸单位的唾液酸寡聚体。用外切神经氨酸酶处理3 H标记的低聚唾液酸，将放射性定量转化为唾液醇，确定脑源性低聚物仅由唾液酸组成。从E. coliK 1能将唾液酸转移到外源性寡聚唾液酸或多聚唾液酸受体上，也能识别大鼠脑细胞膜，进一步证实了多聚唾液酸在大鼠脑中的存在。用E. coliK 1中聚唾液酸的合成缺陷，只能将唾液酸转移到寡聚唾液酸或聚唾液酸的外源受体上。当加入脑细胞膜作为外源受体时，突变体中唾液酸聚合物的合成恢复。
Three prokaryotic-derived probes to identify and study the temporal expression of polysialosyl units in neuronal tissue have been developed. A polyclonal antibody, a bacteriophage-derived endo-neuraminidase, and an Escherichia coli K1 sialyltransferase are all specific for either recognizing or synthesizing poly(sialic acid) containing alpha-2,8-ketosidic linkages. Polysialosyl immunoreactivity with apparent Mr values of 180,000-240,000 was specific for developing neuronal tissue; it was not detected in neonatal liver or kidney or in adult brain tissue. The developmentally regulated disappearance in poly(sialic acid) is consistent with the probes described here recognizing the polysialosyl carbohydrate units of a neuronal cell adhesion molecule (N-CAM). Treatment of brain extracts with a bacteriophage-derived endo-neuraminidase specific for alpha-2,8-linked polysialosyl units abolished the immunoreactivity. The material solubilized by endo-neuraminidase was isolated, reduced with borotritide, and shown to contain oligomers of sialic acid with three to six sialyl units. Treatment of the 3H-labeled oligosialic acid with exo-neuraminidase quantitatively converted the radioactivity to sialitol, establishing that the brain-derived oligomers were composed solely of sialic acid. A membranous sialytransferase from E. coli K1 that can transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid) also recognized rat brain membranes, further substantiating the presence of poly(sialic acid) in rat brain. This conclusion was confirmed by using a mutant of E. coli K1 that was defective in the synthesis of poly(sialic acid) and could only transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid). Sialyl polymer synthesis was restored in the mutant when brain membranes were added as exogenous acceptor.