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从火鸡(吐绶鸡)精浆中分离、鉴定一种Kazal家族蛋白酶抑制剂并进行cDNA测序

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

作者信息

Słowińska Mariola, Olczak Mariusz, Wojtczak Mariola, Glogowski Jan, Jankowski Jan, Watorek Wiesław, Amarowicz Ryszard, Ciereszko Andrzej

机构信息

Department of Semen Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland.

出版信息

Comp Biochem Physiol B Biochem Mol Biol. 2008 Jun;150(2):207-15. doi: 10.1016/j.cbpb.2008.03.003. Epub 2008 Mar 13.

Abstract

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

摘要

火鸡的生殖道和精浆中含有一种丝氨酸蛋白酶抑制剂,这种抑制剂似乎是生殖道所特有的。我们的实验目的是从火鸡精浆和睾丸中分离、鉴定Kazal家族蛋白酶抑制剂并测定其cDNA序列。精浆中含有两种形式的Kazal家族抑制剂:未交配的(Ia),以中等电泳迁移率的抑制剂为代表(睾丸中也存在),以及修饰的(Ib,一种断裂的肽键),以快速迁移率的抑制剂为代表。精浆中的抑制剂通过亲和、离子交换和反相色谱法进行纯化。睾丸抑制剂通过亲和和离子交换色谱法进行纯化。两种精浆抑制剂和睾丸抑制剂的N端埃德曼测序结果相同。该序列用于构建引物并从睾丸中获得cDNA序列。对cDNA序列的分析表明,火鸡蛋白酶抑制剂属于Kazal家族抑制剂(胰腺分泌性胰蛋白酶抑制剂、哺乳动物顶体蛋白酶抑制剂)和钙调蛋白。火鸡精浆Kazal抑制剂属于低分子量抑制剂,其特征在于抑制剂/胰蛋白酶复合物的平衡缔合常数较高。

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