Identification of a Mg2+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms

摘要: Hydrophobic folate-binding proteins (FBPs), which are only 5-10 kDa larger than 40-kDa hydrophilic FBPs, bind significant quantities of Triton X-100 micelles and elute as apparent 160-kDa species on Sephacryl S-200 gel filtration in X-100. Detergent-solubilized placental membranes release a major (greater 95%) FBP well minor folate binding when similarly analyzed. We tested the hypothesis that this recovery predominantly FBPs was mediated by putative hydrophobic FBP-specific protease. When placenta solubilized presence increasing concentrations EDTA, there progressive increase moieties concomitant with decrease FBPs. At 20 mM single recovered could not be detected radioligand or specific radioimmunoassay. The specifically bound radiolabeled folates were immunoprecipitated rabbit anti-40-kDa antiserum. On 15% sodium dodecyl sulfate-polyacrylamide electrophoresis followed transfer to nitrocellulose probing antiserum, electrophoresed 45-kDa species. Detergent studies indicated hydrophobic, thus accounting for molecular weight discrepancy versus electrophoresis. EDTA-mediated inhibition conversion protease reversed dose-dependent manner Mg2+. If is physiologically relevant, it play an important regulatory role transport influencing net number cell surface.