Kinetics and structural analysis of Ebony, a glial protein in the visual system of Drosophila

在节肢动物的视觉系统中，组胺(HA)是视网膜光感受器神经元向下游突触后神经元传递刺激的神经递质。我们提出了这种神经递质在果蝇中的一种循环途径，其中乌木和Tan两种酶以及尚未确定的转运蛋白具有关键功能：来自突触间隙的HA被乌木融合到周围上皮神经胶质细胞中的β-丙氨酸以灭活。转移到光感受器神经元后，β-丙氨酰-组胺或癌(CA)被Tan水解，从而补充HA池以装载囊泡。这种工作模式在很大程度上依赖于乌木的催化效率。为了验证乌木在神经递质失活中起关键作用的循环途径，有必要确定其反应动力学。此外，还将解决酶结构问题，以了解β-丙氨酸的特定募集和多肽键的形成如何与HA快速转运到上皮胶质细胞相一致。作为先决条件，将采用新的表达系统和纯化策略，以克服迄今在制备纯和有活性的乌木蛋白方面的有限能力。

In the visual system of arthropods histamine (HA) is the neurotransmitter for stimulus transmission from retina photoreceptor neurons to downstream postsynaptic neurons. We propose a recycling pathway for this neurotransmitter in Drosophila melanogaster (D. melanogaster) in which the two enzymes Ebony and Tan together with yet to be identified transporters hold key functions: HA from the synaptic cleft is fused to β-alanine by Ebony in the surrounding epithelial glia for inactivation. After transfer into photoreceptor neurons, β-alanyl-histamine or carcinine (CA) is hydrolyzed by Tan thereby replenishing the HA pool for vesicle loading. This working model strongly depends among others on the cata-lytic efficiency of Ebony. To verify the proposed recycling pathway with a key function of Ebony in neurotransmitter inactivation, it is essential to determine its reaction kinetics. Furthermore, enzyme structure will be solved to understand how specific recruitment of β-alanine and peptide bond formation can be performed in concert with fast HA transport into epithelial glia. As a prerequisite new expression systems and purification strategies will be employed that overcome the hitherto limited capability in preparing pure and active Ebony protein.