Adler J E, Schleifer L S, Black I B
Cornell University Medical College, Department of Neurology, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 Feb;86(3):1080-3. doi: 10.1073/pnas.86.3.1080.
Cell membrane contact induces the de novo expression of choline O-acetyltransferase (CAT; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) activity in cultures of virtually pure neonatal rat dissociated sympathetic neurons. To identify molecular mechanisms underlying membrane-associated CAT induction, the responsible membrane component was characterized and partially purified. Substantial CAT-inducing activity was found in membranes from adult rat spinal cord and sensory and sympathetic ganglia. Whole brain membranes demonstrated significantly less activity. CAT induction in sympathetic neurons in response to spinal cord membranes was linear with respect to time, after an initial 6-hr lag. It was also linear with respect to concentrations of spinal cord protein from 2 to 100 micrograms per ml. CAT-inducing activity was extracted from spinal cord membranes by incubation with 100 mM NaCl and was purified approximately 5000-fold by DEAE ion-exchange and gel filtration chromatography. The active factor appears to be an extrinsic protein with an apparent molecular mass of 27 kDa. It is inactivated by trypsin and chymotrypsin but is moderately thermostable, retaining activity at 60 degrees C but not at 90 degrees C.
细胞膜接触可诱导几乎纯的新生大鼠离体交感神经元培养物中胆碱 O - 乙酰转移酶（CAT；乙酰辅酶 A：胆碱 O - 乙酰转移酶，EC 2.3.1.6）活性的从头表达。为了确定膜相关 CAT 诱导的分子机制，对负责的膜成分进行了表征和部分纯化。在成年大鼠脊髓、感觉神经节和交感神经节的膜中发现了大量的 CAT 诱导活性。全脑膜的活性明显较低。在最初 6 小时的延迟后，交感神经元对脊髓膜的 CAT 诱导在时间上呈线性。在每毫升 2 至 100 微克的脊髓蛋白浓度范围内也呈线性。通过与 100 mM NaCl 孵育从脊髓膜中提取 CAT 诱导活性，并通过 DEAE 离子交换和凝胶过滤色谱法纯化约 5000 倍。活性因子似乎是一种表观分子量为 27 kDa 的外在蛋白。它被胰蛋白酶和糜蛋白酶灭活，但具有适度的热稳定性，在 60℃时保留活性，但在 90℃时不保留活性。
