Siebert A, Lottspeich F, Nelson N, Betz H
Abteilung Neurochemie, Max-Planck-Institut für Hirnforschung, Frankfurt/Main, Germany.
J Biol Chem. 1994 Nov 11;269(45):28329-34.
Physophilin is a 36-kDa polypeptide originally identified in synaptic plasma membrane fractions, which binds to synaptic vesicles and has been implicated in vesicle docking and/or exocytosis during neurotransmitter release. Here we report on the purification, amino acid sequence analysis, and subcellular localization of physophilin. Physophilin was enriched from detergent extracts of crude synaptic plasma membranes by a combination of cation exchange and lentil-lectin chromatography. Sequence analysis of peptides generated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that physophilin is identical to the 39-kDa subunit (Ac39) of the vacuolar H(+)-ATPase. This was confirmed further by Western blot analysis with an Ac39-specific antiserum and by vesicle binding assays with recombinant Ac39 protein. Subcellular fractionation showed that Ac39 is enriched in synaptic vesicles, with lesser amounts being present in synaptic plasma membrane fractions. These results argue against a docking role of physophilin/Ac39 in synaptic vesicle exocytosis.
亲物理蛋白是一种最初在突触质膜组分中鉴定出的36 kDa多肽，它与突触小泡结合，并在神经递质释放过程中参与小泡对接和/或胞吐作用。在此，我们报告亲物理蛋白的纯化、氨基酸序列分析及亚细胞定位。通过阳离子交换和扁豆凝集素色谱法相结合，从粗制突触质膜的去污剂提取物中富集亲物理蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后产生的肽段序列分析表明，亲物理蛋白与液泡H(+)-ATP酶的39 kDa亚基（Ac39）相同。用Ac39特异性抗血清进行的蛋白质印迹分析以及用重组Ac39蛋白进行的小泡结合试验进一步证实了这一点。亚细胞分级分离显示，Ac39在突触小泡中富集，在突触质膜组分中的含量较少。这些结果表明亲物理蛋白/Ac39在突触小泡胞吐作用中不具有对接作用。
