Nerve Growth Cones Isolated from Fetal Rat Brain . IV . Preparation of a Membrane Subfraction and Identification of a Membrane Glycoprotein Expressed on Sprouting Neurons
Nerve Growth Cones Isolated from Fetal Rat Brain . IV . Preparation of a Membrane Subfraction and Identification of a Membrane Glycoprotein Expressed on Sprouting Neurons
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从胎鼠大脑中分离出神经生长锥。
DOI：
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
K. H. Pfenninger
中科院分区：
文献类型：
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作者：
L. Ellis;Edith Abreu;K. H. Pfenninger
本研究描述了从分离的神经生长锥颗粒（GCPs）制备膜亚组分（参见Pfenninger，K. H、L.埃利斯，M。P.约翰逊，L. B。Friedman和S. Somlo，1983，Cell，35：573-584）和在该级分中鉴定在神经突生长期间表达的糖蛋白。虽然在粒状（部分破坏）GCP的考马斯亮蓝染色SDS聚丙烯酰胺凝胶中可以看到约40种主要多肽，但从溶解的、洗涤剂渗透的GCP制备的盐水洗膜部分仅含有14%的该蛋白质，并且具有异常简单的七条主要条带的多肽模式。已经产生了从胎鼠脑中分离的GCP膜的单克隆抗体。这些抗体已被筛选差异与突触体从成年大鼠大脑，以确定那些识别抗原的神经突生长过程中选择性表达。一种这样的抗体（称为5 B4）识别发育调节的膜糖蛋白，其在GCP膜中富集并在体外发芽的胎儿神经元中表达。胎脑中的5 B4抗原在SDS聚丙烯酰胺凝胶中迁移为~185255 kD的弥散带，富含唾液酸，由一个小家族的等电变体组成。冻融和神经氨酸酶消化导致天然抗原裂解成两种新的物种，在200和160 kD左右扩散迁移。延长的神经氨酸酶消化使这些条带分别在约180和135 kD处变尖。在成熟脑中，抗体5 B4识别在~140 kD处迁移的稀疏多肽。如以下论文（沃利斯，I.，L. Ellis，K. Suh和K. H. Pfenninger，1985，J. Cell Biol.，101：1990-1998），胎儿抗原特异性地与神经元发芽的区域相关，因此可以用作神经突生长的分子标记。对萌芽神经元的生长锥的生化分析一直受到迄今为止无法分离出足够数量的这些结构的限制。另一个困难是生长锥的结构复杂性：除了质膜，它包含几种类型的细胞器，包括内膜系统（4，10，58，67）。在以前的出版物中，我们已经描述了来自细胞生物学杂志第101卷1985年11月1977-1989 ©洛克菲勒大学出版社的亚细胞部分，其高度富集颗粒，所述颗粒（a）具有神经生长锥的所有细胞学特征（40），（B）与从培养物中显微切割的神经生长锥共纯化（40），和（c）含有神经元特征性磷蛋白的补体（综述参见，例如，参考文献36），包括突触蛋白I及其激酶（11，28）。我们在此描述了神经生长锥颗粒（GCP）的膜亚组分1977的制备，见2017年8月7日jcb.rress.org D ow nladed fomtion（GCM）1。这些膜含有一组简化的多肽，并且可以以足够的量制备（100-#g a m o n t s），用于聚合物和B o d i e m o n a l a n t i e n（m A B s）阿加t he ir c om p o n e t s .该等债券的发行乃基于以下基准。在执行B操作期间，由于它将单个单元中的单个单元区分为不同的位置，因此，最后，与具有适当预处理目标单元的同步捕获相关联。对增长规格分析的一个重要补充是，在特定的新开发阶段，例如，在一个实施例中，在G C P s与S Y N A P T O S O M S中出现的并发症（来自硬拷贝的最大并发症）。G C P和S的子元素数量的可用性不稳定性对于某些模型来说，使用这些子元素作为A B的一般规则是可行的。如果要选择一个B对象，则必须通过对B对象进行筛选来重新识别已重新定义的对象。在一个即将被淘汰的B L的地方，我们将在系统中描述一个简单的扩展，以使B U不在G C P中（参见。参考文献62）。在前期研究中，我们描述了一个A B b，它在一个非常小的系统中实现了对G C P m e m B r a n e s的高度可调节性，外消旋离子。这种独特的表现形式与多种聚合物混合物的微生物降解能力有关。这些发现的预浸提液在吸收法中得到了充分的B e预浸提（62，63）。M A T E R I A L S A N D M E T O D S
This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573-584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While ~40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a saltwashed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of ~185255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezingthawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at ~140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth. The biochemical analysis of growth cones of sprouting neurons has been limited by the inability heretofore to isolate these structures in sufficient quantity. A further difficulty is the structural complexity of the growth cone: in addition to the plasma membrane, it contains several types of organelle including endomembrane systems (4, 10, 58, 67). In previous publications, we have described a subcellular fraction from THE JOURNAL OF CELL BIOLOGY VOLUME 101 NOVEMBER 1985 1977-1989 © The Rockefeller University Press • 0021-9525/85[11/1977/13 $1.00 fetal rat brain highly enriched in particles that (a) have all the cytological characteristics of nerve growth cones (40), (b) copurify with nerve growth cones microdissected from cultures (40), and (c) contain a complement of phosphoproteins characteristic of neurons (for review see, e.g., reference 36), including synapsin I and its kinase (11, 28). We describe here the preparation of a membrane subfrac1977 on A uust 7, 2017 jcb.rress.org D ow nladed fom tion (GCM) l of the nerve growth cone particle (GCP). These membranes contain a simplified set of polypeptides and can be p r e p a r e d in suf f ic ien t q u a n t i t y (100-#g a m o u n t s ) for t he g e n e r a t i o n o f po lyc lona l a n d m o n o c l o n a l a n t i b o d i e s ( m A b ' s ) aga ins t t he i r c o m p o n e n t s . T h e p r e p a r a t i o n o f such a n t i b o d i e s was b a s e d o n the fo l lowing ra t iona le . D u r i n g e m b r y o g e n e s i s , t he n e u r o n u n d e r g o e s d r a m a t i c c h a n g e s in p h e n o t y p e as it d i f f e ren t i a t e s f r o m a d i v i d i n g n e u r o e p i t h e l i a l cell i n t o a pos t mi to t i c , s p r o u t i n g n e u r o n and , f inal ly, i n to a n e u r o n wi th s y n a p t i c c o n n e c t i o n s w i th t h e a p p r o p r i a t e ta rge t cells. O n e e x p e r i m e n t a l a p p r o a c h to t h e ana lys i s o f g rowth spec i f i c m o l ecu les is t h e i m m u n o c h e m i c a l c o m p a r i s o n o f c o m p o n e n t s exp res sed d u r i n g specif ic s tages o f n e u r o n a l d e v e l o p m e n t , e.g., c o m p o n e n t s p r e s e n t in G C P s vs. s y n a p t o s o m e s ( the i r m a t u r e c o u n t e r p a r t f r o m a d u l t bra in) . T h e avai labi l i ty o f subs t an t i a l q u a n t i t i e s o f G C P s a n d s y n a p t o s o m e s m a k e s it feasible to use t hese subce l lu l a r f r ac t ions as a n t i g e n s for t h e g e n e r a t i o n o f m A b ' s . O n e can t h e n select t he a n t i b o d i e s t ha t r ecogn ize d e v e l o p m e n t a l l y r egu la t ed n e u r o n a l an t i g en s by s c r een ing d i f fe ren t ia l ly wi th b o t h f rac t ions . In a r e p o r t to be p u b l i s h e d shor t ly we will de sc r ibe a n an t i g en exp re s sed in s y n a p t o s o m e s b u t n o t in G C P s (cf. r e f e rence 62). In t h e p r e s e n t s tudy , we desc r ibe a m A b w h i c h r ecogn izes a d e v e l o p m e n t a l l y r egu la t ed n e u r o n a l a n t i g e n o f G C P m e m b r a n e s t ha t is p r e s e n t o n l y in very smal l a m o u n t s in s y n a p t o s o m e f rac t ions . T h e an t i g en exh ib i t s b i o c h e m i c a l p r o p e r t i e s c o n s i s t e n t w i th t h o s e o f a po lys ia ly la ted m e m b r a n e g lycop ro t e in . P r e l i m i n a r y r ep o r t s o f t hese f i nd ings have b e e n p r e s e n t e d in ab s t r ac t f o r m (62, 63). M A T E R I A L S A N D M E T H O D S
从胚胎脑膜纯化的神经细胞粘附分子的化学表征。
DOI：
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发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Hoffman,S;Sorkin,BC;White,PC;Brackenbury,R;Mailhammer,R;Rutishauser,U;Cunningham,BA;Edelman,GM
通讯作者：
Edelman,GM