大鼠肝细胞膜丝氨酸内肽酶的部分纯化及特性研究

Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes.

作者信息

Guenet L, Gueble-Val F, Blayau M, Le Treut A, Le Gall J Y

出版信息

Biochim Biophys Acta. 1986 Apr 11;881(2):258-67. doi: 10.1016/0304-4165(86)90012-7.

Abstract

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: solubilization with N-lauroylsarcosine; Ultrogel AcA-34 chromatography; CM Affi-Gel blue chromatography; agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H-D-Val-Leu-Arg-p-nitroanilide, reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH optimum between 8.0 and 9.0 and was inhibited by D-Phe-L-Phe-L-Arg-CH2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [3H]DFP-labelled protein electrophoresis.

摘要

采用四步法从大鼠肝细胞膜中部分纯化了一种丝氨酸内肽酶

用N-月桂酰肌氨酸溶解；进行Ultrogel AcA - 34柱层析；CM Affi - Gel蓝柱层析；琼脂糖 - 大豆胰蛋白酶抑制剂柱层析。发现该酶可水解酪蛋白和各种生色肽底物；对H - D - Val - Leu - Arg - p - 硝基苯胺的活性最高，据报道该底物是人类腺体激肽释放酶的特异性底物。该酶对热敏感，最适pH在8.0至9.0之间，并且受到D - Phe - L - Phe - L - Arg - CH2Cl、抑肽酶、二异丙基氟磷酸酯（DFP）、大豆胰蛋白酶抑制剂、苯甲基磺酰氟、亮抑酶肽、抗蛋白酶和二硫苏糖醇的抑制。通过Ultrogel AcA - 34柱层析以及对[3H]DFP标记的蛋白质电泳进行放射自显影测定，这种肝细胞膜蛋白酶的表观分子量约为30000。