ACTIVITY AND BIOSPECIFICITY OF PROTEOLYZED FORMS AND DIMERIC COMBINATIONS OF RECOMBINANT HUMAN AND MURINE NERVE GROWTH-FACTOR
ACTIVITY AND BIOSPECIFICITY OF PROTEOLYZED FORMS AND DIMERIC COMBINATIONS OF RECOMBINANT HUMAN AND MURINE NERVE GROWTH-FACTOR
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DOI：
10.1111/j.1471-4159.1992.tb11030.x
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发表时间：
1992-11-01
影响因子：
4.7
通讯作者：
GORRELL, A
中科院分区：
文献类型：
--
作者：
BURTON, LE;SCHMELZER, CH;GORRELL, A
纯化的重组人神经生长因子（rhNGF）和颌下腺衍生的小鼠神经生长因子（muNGF），其特征在于氨基酸组成，聚丙烯酰胺凝胶电泳（PAGE），反相高效液相色谱（RP-HPLC），和高效离子交换色谱（HPIEC）。120个残基形式的rhNGF的N和C末端的有限胰蛋白酶消化产生109个残基的种类（10- 118）。先前观察到的天然鼠类似物的这种变体，muNGF缺乏前八个N-末端氨基酸，也被分离为同源二聚体。使用HPIEC纯化两种物质，并通过氨基酸分析、N-末端序列、PAGE和RP-HPLC分析进行表征。在以下部分或全部NGF生物学测定中对四种同二聚体物种中的每一种进行了评价：鸡背根和交感神经节测定以及大鼠嗜铬细胞瘤12细胞系神经突延伸测定。rhNGF和muNGF的118个残基的同源二聚体版本显示出相当的生物活性，而N末端修饰的分子呈现出50至100倍的活性降低。利用HPIEC，我们已经研究了单体形式的任何两个rhNGF的同质二聚体物种重组的能力。我们已经表明，不仅可以通过重组所有先前描述的物种形成二聚体，但种间二聚体可以创建之间的muNGF和rhNGF。
Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland-derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed-phase HPLC (RP-HPLC), and high-performance ion-exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120-residue form of rhNGF produced a species of 109 residues (10-1 18). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N-terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N-terminal sequence, PAGE, and RP-HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma 12 cell line neurite extension assay. The 118-residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal-modified molecules presented activity reduced by 50- to 100-fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.