Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 °C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila.

中文翻译：

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家蝇消化胰凝乳蛋白酶的特性，分泌机制及其与果蝇同源物的关系。

家蝇幼虫在后中肠（PMG）中表现出两种不同的消化性胰凝乳酶活性：一种主要的可溶性活动存在于管腔中，另一种次要的存在于细胞膜组分中。在存在和不存在10 mM Triton X-100的情况下，可溶性和膜结合型胰凝乳蛋白酶活性均具有不同的热灭活半衰期（46°C），表明它们是两种不同的分子种类。在SDS-PAGE中纯化的可溶性胰凝乳蛋白酶活性的最适pH为7.4，分子量为28 kDa。它不会切割短的底物（例如Suc-F-MCA），而会切割较长的底物（例如Suc-AAPF-MCA），而对Phe而不是Tyr和Leu残基具有主要特异性（kcat / Km）。凝胶中的活性显示出针对S-AAPF-MCA的独特条带，其迁移与纯化的胰凝乳蛋白酶相同。在大肠杆菌（DE3）Star中对一种胰凝乳蛋白酶原样序列（MdChy1）进行了测序，克隆和重组表达。通过RT-PCR观察，MdChy1在近端后中肠（PMG1）中表达。其他胰凝乳蛋白酶基因的表达分析揭示了在前中肠（AMG）和PMG表达的基因。使用抗MdChy1抗血清对家蝇中肠组织进行的蛋白质印迹显示，来自AMG和PMG的样品中有一条条带与重组和纯化的酶共同迁移。在小囊泡（因此表明胞吐作用）以及AMG和PMG内腔中发现了与Mdchy1对应的免疫金标记，证实了两组相似的胰凝乳蛋白酶。通过焦磷酸测序制备的家蝇支原体AMG和整个中肠的转录组揭示了41种独特的胰凝乳蛋白酶样酶序列（其中19种可能起作用），从中高度表达MdChy1。果蝇和M. domestica胰凝乳蛋白酶样序列的系统发育重建表明，胰凝乳蛋白酶基因在Musca和果蝇的进化分离之前先扩展。