Ruppert C, Sandrasagra A, Anton B, Evans C, Schweitzer E S, Tobin A J
Department of Biology, University of California, Los Angeles 90024-1606.
J Neurochem. 1993 Aug;61(2):768-71. doi: 10.1111/j.1471-4159.1993.tb02186.x.
We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD65, GAD67, or both). Individual clones of engineered Rat-1 cells make the appropriate GAD mRNAs and GAD polypeptides, show GAD enzymatic activity, and make GABA. Clones expressing GAD65 had higher enzymatic activity than those expressing GAD67. As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD65 is more responsive to added pyridoxal phosphate than that of GAD67. Immunostaining for both GADs is scattered throughout the cytoplasm. GAD65 immunostaining is less homogeneous than that of GAD67 and also appears to be associated with the surfaces of large vesicle-like structures. Cells expressing GAD65 and GAD67 showed similar immunostaining patterns with anti-GABA antibodies and contained substantial amounts of GABA (ranging from 7 to 18 pmol of GABA/10(6) cells), which was roughly proportional to their levels of GAD activity. GABA is released from the engineered cells into the surrounding medium under resting conditions, suggesting that cells programmed with GAD cDNAs might serve as effective sources of GABA in cell transplantation experiments.
我们使用了逆转录病毒cDNA表达系统来驱动不同形式的谷氨酸脱羧酶（GAD65、GAD67或两者）的表达。工程化的大鼠-1细胞的各个克隆产生适当的GAD mRNA和GAD多肽，显示出GAD酶活性，并产生GABA。表达GAD65的克隆比表达GAD67的克隆具有更高的酶活性。与脑GAD以及工程细菌中产生的GAD情况一样，GAD65的酶活性比GAD67对添加的磷酸吡哆醛更敏感。两种GAD的免疫染色分散在整个细胞质中。GAD65的免疫染色不如GAD67均匀，并且似乎也与大的囊泡状结构的表面相关。表达GAD65和GAD67的细胞用抗GABA抗体显示出相似的免疫染色模式，并且含有大量的GABA（范围为7至18 pmol GABA/10⁶个细胞），这与其GAD活性水平大致成比例。在静息条件下GABA从工程化细胞释放到周围培养基中，这表明用GAD cDNA编程的细胞可能在细胞移植实验中作为有效的GABA来源。
