Isolation and characterization of a folate receptor-directed metalloprotease from human placenta.
Glycosyl-phosphatidylinositol-anchored hydrophobic placental folate receptors (PFRs), which have an important functional role in maternal-to-fetal transplacental folate transport, can be converted to soluble hydrophilic forms by a placental metalloprotease. Using a Triton X-114 temperature-induced phase separation assay to monitor enzyme-mediated conversion of radiolabeled hydrophobic PFR into hydrophilic PFR, a metalloenzyme was isolated to apparent homogeneity from Triton X-114-solubilized human placenta using concanavalin A-Sepharose and reverse-phase high performance liquid chromatography (HPLC) as major purification steps. The purified hydrophobic enzyme eluted as a single protein peak on reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis revealed a single 63,000 M(r) species, which was reduced to 58,000 M(r) following deglycosylation, findings comparable with amino acid analysis (M(r) approximately 59,000). The metalloenzyme was activated by Mg2+, Zn2+, Mn2+, and Ca2+, optimally at physiologic pH; it also exhibited EDTA-sensitive endoproteolytic cleavage of [3H]leucine-labeled full-length nascent PFR polypeptide generated in vitro in the absence of microsomes. Rabbit polyclonal anti-metalloprotease antiserum specifically immunoprecipitated 125I-metalloprotease and recognized cross-reacting moieties on plasma membranes of normal human hematopoietic progenitor cells and human cervical carcinoma cells, both of which also express FR.
Yang
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Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Mackins
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JY
Li
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Antony
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AC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011817:Rabbits; D011956:Receptors, Cell Surface; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000201:enzymology
D000596:Amino Acids; D000906:Antibodies; D002352:Carrier Proteins; D002413:Cations, Divalent; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D003208:Concanavalin A; D008666:Metalloendopeptidases