McMorris F A, Kim S U, Sprinkle T J
Brain Res. 1984 Jan 30;292(1):123-31. doi: 10.1016/0006-8993(84)90896-5.
We investigated whether the membrane-associated myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), is localized primarily inside the cell or exposed on the cell surface of rat oligodendrocytes and rat C6 glioma cells. Determinations were made by enzyme assays of intact, viable cells vs cells broken by freezing and thawing. Assay of both oligodendrocytes and C6 cells showed that the great majority of the CNP activity was localized inside the cells. Oligodendrocytes were also tested by immunofluorescence staining of unfixed, living cells whose membranes had been made permeable to antibody by fixation. Fixed oligodendrocytes showed intense fluorescence when incubated with rabbit anti-CNP antiserum and fluorescein-conjugated second antibody whereas unfixed cells were not stained. We then tested the possible influence on CNP localization of 3 conditions known to increase CNP specific activity: maturation of oligodendrocytes in vitro during a period when CNP specific activity increases 8-fold or more; growth of C6 cultures to high cell density; and induction of CNP activity in oligodendrocytes and C6 cells by dibutyryl cyclic AMP. Under all conditions, most CNP activity was intracellular. These results show that both the catalytic and major antigenic sites of CNP are localized primarily inside the cell, and suggest an intracellular role for CNP in oligodendrocytes. The results with C6 cells also show that these cells resemble oligodendrocytes with respect to CNP localization.
我们研究了膜相关髓磷脂酶2',3'-环核苷酸3'-磷酸水解酶（CNP；EC 3.1.4.37）主要是定位于大鼠少突胶质细胞和大鼠C6胶质瘤细胞的细胞内还是暴露于细胞表面。通过对完整活细胞与经冻融破碎的细胞进行酶活性测定来进行判断。对少突胶质细胞和C6细胞的测定均表明，绝大多数CNP活性定位于细胞内。还通过对未固定的活细胞进行免疫荧光染色来检测少突胶质细胞，这些活细胞的细胞膜已通过固定处理使其对抗体具有通透性。当用兔抗CNP抗血清和荧光素偶联的二抗孵育时，固定的少突胶质细胞显示出强烈的荧光，而未固定的细胞未被染色。然后，我们测试了已知可增加CNP比活性的3种条件对CNP定位的可能影响：在体外少突胶质细胞成熟期间，CNP比活性增加8倍或更多；将C6培养物培养至高细胞密度；以及用二丁酰环磷腺苷诱导少突胶质细胞和C6细胞中的CNP活性。在所有条件下，大多数CNP活性都在细胞内。这些结果表明，CNP的催化位点和主要抗原位点主要定位于细胞内，并提示CNP在少突胶质细胞中具有细胞内作用。对C6细胞的研究结果还表明，这些细胞在CNP定位方面类似于少突胶质细胞。
