Isolation and characterization of a folate receptor-directed metalloprotease from human placenta

Xiao Yan Yang, Janet Y. Mackins, Qing Jun Li, Aśok C. Antony

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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) ~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.

Original languageEnglish (US)
Pages (from-to)11493-11499
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number19
DOIs
StatePublished - Jun 4 1996

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Folate Receptor 2
Metalloproteases
Folic Acid
Placenta
High performance liquid chromatography
Reverse-Phase Chromatography
High Pressure Liquid Chromatography
Glycosylphosphatidylinositols
Enzymes
Cell membranes
Hematopoietic Stem Cells
Microsomes
Electrophoresis
Edetic Acid
Leucine
Phase separation
Purification
Immune Sera
Polyacrylamide Gel Electrophoresis
Assays

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Isolation and characterization of a folate receptor-directed metalloprotease from human placenta. / Yang, Xiao Yan; Mackins, Janet Y.; Li, Qing Jun; Antony, Aśok C.

In: Journal of Biological Chemistry, Vol. 271, No. 19, 04.06.1996, p. 11493-11499.

Research output: Contribution to journalArticle

Yang, Xiao Yan ; Mackins, Janet Y. ; Li, Qing Jun ; Antony, Aśok C. / Isolation and characterization of a folate receptor-directed metalloprotease from human placenta. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 19. pp. 11493-11499.
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