Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex
Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex
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小牛脑皮质突触体膜中唾液酸转移酶活性的存在
DOI：
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发表时间：
1980
影响因子：
4.7
通讯作者：
G. Tettamanti
中科院分区：
文献类型：
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作者：
A. Preti;A. Fiorilli;A. Lombardo;L. Caimi;G. Tettamanti
利用小牛脑皮层，研究了在神经末梢周围的质膜（突触体膜）中可能发生的唾液转移酶活性。突触体膜是通过一种改进的程序制备的，该程序提供：(a)“神经末梢部分”由至少85%保存完好的神经末梢组成，仅含有少量细胞内起源的膜；(b)“突触体膜组分”携带大量的真正的质膜标记物（Na+ K+ atp酶，5′-核苷酸酶，唾液酸酶，神经节苷脂），其比活性值比“神经末梢组分”高4到5倍，并且非常少量的脑苷亚硫酸盐转移酶，高尔基体标记物，以及其他细胞膜标记物（鱼藤酮不敏感的NADH和NADPH）；细胞色素c还原酶)，其具体活性分别是“神经末梢部分”的0.5倍和0.7倍。因此，所使用的突触体膜的制备具有质膜的特性，并且对细胞内源膜的污染可以忽略不计。唾液基转移酶在主要脑亚细胞部分（微粒体、P2部分、神经末段、线粒体）的活性分布与硫胺素焦磷酸酶（已知与高尔基体和质膜有关的酶）和乙酰胆碱酯酶（已知与细胞内或质膜有关的酶）的活性分布最为相似。唾液基转移酶活性在“突触体膜组分”（即“神经末梢组分”）中的富集与真正的质膜标记物所显示的几乎相同。所有这些都与小牛大脑皮层唾液转移酶有两个不同的亚细胞位置的假设相一致：一个在细胞内结构的水平，最有可能是高尔基体（如其他作者所描述的），另一个在突触体质膜。建立了突触体膜结合唾液基转移酶的基本性质（pH最适、V/S、V/t和V/蛋白关系）和洗涤需求。外源受体、乳糖神经酰胺和ds - fetuin的酶活性最高。以乳糖神经酰胺和ds - fetuin为受体底物时，CMP‐NeuNAc的Km值不同（分别为0.57和0.135 mm）；酶作用于糖脂受体的热稳定性高于作用于糖蛋白受体的热稳定性；使用来自糖脂受体的糖蛋白时，洗涤剂的效果不同；没有观察到乳糖神经酰胺和ds - fetuin之间的竞争。因此，突触体膜携带至少两种不同的唾液转移酶活性：一种作用于乳糖神经酰胺（和糖脂受体），另一种作用于ds - fetuin（和糖蛋白受体）。神经节苷脂GM3被认为是突触体膜结合唾液基转移酶活性作用于乳糖神经酰胺作为受体底物的产物。
The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide as acceptor substrate.