Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
Insect Biochem Mol Biol. 2012 Jul;42(7):482-90. doi: 10.1016/j.ibmb.2012.03.005.
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.
家蝇幼虫的后中肠 (PMG) 中有两种不同的消化糜蛋白酶活性：一种是腔可溶性主要活性，另一种是细胞膜部分的次要活性。在有或没有 10 mM Triton X-100 的情况下，可溶性和膜结合的糜蛋白酶活性的热失活半衰期 (46°C) 不同，表明它们是两种不同的分子种类。纯化的可溶性糜蛋白酶活性在 SDS-PAGE 中的 pH 最适值为 7.4，分子量为 28 kDa。它不切割短底物，如 Suc-F-MCA，而更喜欢长底物，如 Suc-AAPF-MCA，对 Phe 的一级特异性 (kcat/Km) 而不是 Tyr 和 Leu 残基。胶内活性显示出与纯化的糜蛋白酶相同迁移率的针对 S-AAPF-MCA 的独特带。一个糜蛋白酶原样序列 (MdChy1) 被测序、克隆并在大肠杆菌 (DE3) Star 中重组表达。MdChy1 通过 RT-PCR 在近端后中肠 (PMG1) 中表达。对其他糜蛋白酶基因的表达分析显示，基因在前中肠 (AMG) 和 PMG 中表达。用抗 MdChy1 抗血清对 M. domestica 中肠组织进行 Western blot 分析显示，AMG 和 PMG 样本中均有一条带，与重组和纯化酶共迁移。在家蝇的 AMG 和 PMG 的小泡 (因此表明胞吐作用) 和腔中发现了对应于 Mdchy1 的免疫金标记，证实了存在两组相似的糜蛋白酶。通过焦磷酸测序制备的 M. domestica AMG 和整个中肠的转录组揭示了 41 种独特的糜蛋白酶样酶序列 (19 种可能具有功能)，其中 MdChy1 高度表达。黑腹果蝇和 M. domestica 糜蛋白酶样序列的系统发育重建表明，糜蛋白酶基因在 Musca 和 Drosophila 进化分离之前就已经扩张。
