Deperthes D, Chapdelaine P, Tremblay R R, Brunet C, Berton J, Hébert J, Lazure C, Dubé J Y
Laboratory of Hormonal Bioregulation, CHUL Research Center, Sainte-Foy, Quebec, Canada.
Biochim Biophys Acta. 1995 Dec 14;1245(3):311-6. doi: 10.1016/0304-4165(95)00118-2.
To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.
为了证明激肽释放酶hK2在人前列腺和精浆中的存在，我们使用了针对重组hK2融合蛋白的小鼠单克隆抗体（MAb）。使用其中一种MAb 9D5，我们在前列腺细胞溶质和精浆中检测到了几个主要的22 kDa免疫反应斑点以及次要的31 kDa和55 kDa免疫反应斑点。对精浆蛋白进行离子交换和免疫亲和层析后，22 kDa免疫反应蛋白与55 kDa和75 kDa蛋白一起被分离出来。通过对氨基末端氨基酸测序，分别在22 kDa和55 kDa条带中鉴定出了hK2和蛋白C抑制剂的片段。此外，在一维和二维凝胶上用两种不同的抗hK2单克隆抗体和一种抗蛋白C抑制剂多克隆抗体进行免疫印迹实验表明，在巯基乙醇存在的情况下，主要的55 kDa和75 kDa条带是共价的hK2-蛋白C抑制剂复合物，其中包含全长hK2链或仅其羧基末端片段。这些结果首次证明了激肽释放酶hK2的存在，并表明蛋白C抑制剂可能在精浆中调节其活性。
