Identification, Isolation, and Characterization of a 41-Kilodalton Protein from Rat Germ Cell-Conditioned Medium Exhibiting Concentration-Dependent Dual Biological Activities

In this report, we describe the purification of a novel protease with dual biological actions from germ cell-conditioned medium (GCCM) where germ cells were isolated from adult rat testes using a mechanical procedure. Using multiple HPLC columns and two sequential high performance electrophoresis chromatography steps in association with an [125I]-collagen film assay to detect protease activity, a 41-kDa polypeptide (41-kDa-P) was purified to apparent homogeneity from GCCM. Partial N-terminal amino acid sequence analysis of the purified protein revealed a sequence of NH2-KYEFYEIXLL that, when compared with the existing database at Protein Identification Resource (PIR), GenBank, and BLAST revealed that this is a unique protein. The purified protein, when incubated with [125I]-testin, a Sertoli cell secretary product that is localized at the intertesticular cell junction and is resistant to tryptic digest, was found capable of hydrolyzing testin dose dependently. The proteolysis of [125I]-testin by this 41-kDa protein was inhibited by α2-macroglobulin (a Sertoli cell secretary product) also in a dose-dependent manner. A study on the interactions between different classes of protease inhibitors and the purified 41-kDa protein revealed that it is a serine protease. At doses ranging between 0.5 and 50 ng/ml, 41-kDa-P induced a dose-dependent inhibition of Sertoli cell secretory function using testin and clusterin as markers without any apparent proteolytic activity. However, at doses greater than 0.5 μg/ml, 41-kDa-P was found to cleave [125I]- collagen and [125I]-testin at physiological pH, indicating that this 41- kDa protein has dual biological activities whose primary action is concentration dependent. In view of the biological activities of this protease, it is postulated that this protein may be involved in facilitating germ cell migration in the epithelium.published_or_final_versio