Landa C A, Defilpo S S, Maccioni H J, Caputto R
J Neurochem. 1981 Oct;37(4):813-23. doi: 10.1111/j.1471-4159.1981.tb04466.x.
Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[3H]N-acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H]sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 M-KCl, 125 mM-EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of th neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labelled in vivo by an intracerebral injection of N-[3H]acetylmannosamine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with behaviour of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.
通过将大鼠脑匀浆的神经元胞体与CMP - [³H]N - 乙酰神经氨酸一起孵育，获得了标记的神经节苷脂和糖蛋白。在一个亚细胞组分中观察到最高程度的标记，该组分对几种神经节苷脂糖基转移酶也显示出最高的比活性。在用1M - KCl、125mM - EDTA处理、反复冻融或控制超声处理后，该组分的[³H]唾液酸糖缀合物仍与膜结合，但在脱氧胆酸钠（DOC）浓度高到足以溶解胆碱磷脂时被溶解。这些标记的内源性神经节苷脂和糖蛋白中约75%的对神经氨酸酶敏感的唾液酸残基受到添加的神经氨酸酶或链霉蛋白酶或两种酶同时添加的作用的保护。用高离子强度或EDTA对膜进行预处理不会消除这种保护作用，但超声处理或低浓度的DOC会消除这种保护作用。通过脑内注射N - [³H]乙酰甘露糖胺在体内标记的神经元胞体膜组分的神经节苷脂中，50%至80%的对神经氨酸酶敏感的唾液酸残基在注射后3小时也受到添加的神经氨酸酶作用的保护。添加DOC会消除这种保护作用。与该神经元胞体组分的标记糖缀合物的行为相反，完整突触体中的神经节苷脂和唾液酸糖蛋白可被神经氨酸酶作用。有人提出，大多数神经节苷脂和唾液酸糖蛋白作为神经元胞体膜组分的内在成分被唾液酸化，并且在通过轴突运输并整合到突触质膜的过程的某个阶段，它们对添加酶的可及性发生了变化。
