McManaman J L, Crawford F G, Stewart S S, Appel S H
Department of Neurology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1988 Apr 25;263(12):5890-7.
Extracts of rat skeletal muscle contain neurotrophic factors which stimulate the development of choline acetyltransferase in embryonic day 14 rat spinal cord cultures. The trophic activity does not bind heparin-Sepharose or lectin affinity columns. However, mild acid treatment separates the trophic activity into soluble and insoluble fractions. The acid-insoluble activity has been purified 5000-fold to apparent homogeneity using preparative sodium dodecyl sulfate gel electrophoresis to achieve final purification. The purified factor migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent molecular mass of 20 kDa and a pI of 4.8. The activity and apparent molecular weight of the purified factor are unaltered by treatment with reducing agents or incubation in acidic conditions. Activity, however, is destroyed by heating or protease treatment. Thus, the factor appears to be a single polypeptide without significant levels of glycosylation or charge microheterogeneity. These results represent the first purification of a neurotrophic factor from skeletal muscle. The physical properties and amino acid composition of this factor differ from those of nerve growth factor and heparin-binding growth factors, as well as from the neurotrophic factor from heart cell conditioned medium which induces cholinergic development in sympathetic neurons.
大鼠骨骼肌提取物含有神经营养因子，可刺激胚胎第14天大鼠脊髓培养物中胆碱乙酰转移酶的发育。这种营养活性不与肝素-琼脂糖或凝集素亲和柱结合。然而，温和的酸处理可将营养活性分离为可溶和不溶部分。使用制备型十二烷基硫酸钠凝胶电泳进行最终纯化，酸不溶性活性已纯化5000倍，达到明显的均一性。纯化后的因子在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和等电聚焦上迁移为单一条带，表观分子量为20 kDa，pI为4.8。用还原剂处理或在酸性条件下孵育后，纯化因子的活性和表观分子量均未改变。然而，加热或蛋白酶处理会破坏活性。因此，该因子似乎是一种单一的多肽，没有明显水平的糖基化或电荷微不均一性。这些结果代表了首次从骨骼肌中纯化出神经营养因子。该因子的物理性质和氨基酸组成不同于神经生长因子和肝素结合生长因子，也不同于来自心脏细胞条件培养基的神经营养因子，后者可诱导交感神经元中的胆碱能发育。
