Kinetic analysis, isolation, and characterization of hydrophilic folate-binding proteins released from chorionic villi cultured under serum-free conditions

Rama S. Verma, Asok Antony

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8 Citations (Scopus)

Abstract

Full term placental chorionic villi cultured for 7 days in serum-free medium released hydrophilic folate-binding proteins (S-FBP(PCM)) into the conditioned medium; in contrast, hydrophobic FBPs were the only form recovered from chorionic villi. Kinetic studies revealed that (i) S-FBP(PCM) was maximally released by the 3rd day, and this was associated with a proportionate decrease in hydrophobic FBPs; (ii) although cycloheximide inhibited de novo synthesis of [35S]methionine-labeled hydrophobic FBPs and S-FBP(PCM) by >90%, unlabeled net S-FBP(PCM) release was only inhibited by 50%; (iii) EDTA markedly inhibited release of S-FBP(PCM) which was accompanied by a proportionate increase in hydrophobic FBPs; (iv) EDTA effects were completely reversed by 5-fold molar excesses of Mg2+ which led to a 50-fold greater release of S-FBP(PCM) compared to EDTA alone; (v) whereas Mg2+ alone stimulated S-FBP(PCM) release 4-fold greater than basal conditions, addition of cycloheximide to Mg2+ suppressed (by 4-fold) the expected increase observed with Mg2+ alone. Biochemical analyses of isolated S-FBP(PCM) revealed similarities to hydrophobic FBPs with respect to the ligand-binding domain and epitopes but differed in detergent-binding characteristics; furthermore, amino acid and carbohydrate analysis revealed a lower Mr = 25,500 with 12% carbohydrate. Based on kinetic analysis of S-FBP(PCM) release from chorionic villi-associated hydrophobic FBPs as well as structural analysis of S-FBP(PCM), these data continue to support the hypothesis that (α) a significant amount of maternal and probably fetal serum hydrophilic FBPs originate from placental hydrophobic FBPs, and (b) the endogenous hydrophobic FBP-directed Mg2+-dependent placental protease plays a major role in their release.

Original languageEnglish
Pages (from-to)12522-12535
Number of pages14
JournalJournal of Biological Chemistry
Volume266
Issue number19
StatePublished - 1991

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Chorionic Villi
Pulse code modulation
Folic Acid
Carrier Proteins
Edetic Acid
Kinetics
Cycloheximide
Serum
Carbohydrates
Protein S
Serum-Free Culture Media
Conditioned Culture Medium
Methionine
Detergents
Epitopes
Peptide Hydrolases
Mothers
Ligands
Amino Acids
Structural analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Kinetic analysis, isolation, and characterization of hydrophilic folate-binding proteins released from chorionic villi cultured under serum-free conditions",
abstract = "Full term placental chorionic villi cultured for 7 days in serum-free medium released hydrophilic folate-binding proteins (S-FBP(PCM)) into the conditioned medium; in contrast, hydrophobic FBPs were the only form recovered from chorionic villi. Kinetic studies revealed that (i) S-FBP(PCM) was maximally released by the 3rd day, and this was associated with a proportionate decrease in hydrophobic FBPs; (ii) although cycloheximide inhibited de novo synthesis of [35S]methionine-labeled hydrophobic FBPs and S-FBP(PCM) by >90{\%}, unlabeled net S-FBP(PCM) release was only inhibited by 50{\%}; (iii) EDTA markedly inhibited release of S-FBP(PCM) which was accompanied by a proportionate increase in hydrophobic FBPs; (iv) EDTA effects were completely reversed by 5-fold molar excesses of Mg2+ which led to a 50-fold greater release of S-FBP(PCM) compared to EDTA alone; (v) whereas Mg2+ alone stimulated S-FBP(PCM) release 4-fold greater than basal conditions, addition of cycloheximide to Mg2+ suppressed (by 4-fold) the expected increase observed with Mg2+ alone. Biochemical analyses of isolated S-FBP(PCM) revealed similarities to hydrophobic FBPs with respect to the ligand-binding domain and epitopes but differed in detergent-binding characteristics; furthermore, amino acid and carbohydrate analysis revealed a lower Mr = 25,500 with 12{\%} carbohydrate. Based on kinetic analysis of S-FBP(PCM) release from chorionic villi-associated hydrophobic FBPs as well as structural analysis of S-FBP(PCM), these data continue to support the hypothesis that (α) a significant amount of maternal and probably fetal serum hydrophilic FBPs originate from placental hydrophobic FBPs, and (b) the endogenous hydrophobic FBP-directed Mg2+-dependent placental protease plays a major role in their release.",
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AU - Verma, Rama S.

AU - Antony, Asok

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N2 - Full term placental chorionic villi cultured for 7 days in serum-free medium released hydrophilic folate-binding proteins (S-FBP(PCM)) into the conditioned medium; in contrast, hydrophobic FBPs were the only form recovered from chorionic villi. Kinetic studies revealed that (i) S-FBP(PCM) was maximally released by the 3rd day, and this was associated with a proportionate decrease in hydrophobic FBPs; (ii) although cycloheximide inhibited de novo synthesis of [35S]methionine-labeled hydrophobic FBPs and S-FBP(PCM) by >90%, unlabeled net S-FBP(PCM) release was only inhibited by 50%; (iii) EDTA markedly inhibited release of S-FBP(PCM) which was accompanied by a proportionate increase in hydrophobic FBPs; (iv) EDTA effects were completely reversed by 5-fold molar excesses of Mg2+ which led to a 50-fold greater release of S-FBP(PCM) compared to EDTA alone; (v) whereas Mg2+ alone stimulated S-FBP(PCM) release 4-fold greater than basal conditions, addition of cycloheximide to Mg2+ suppressed (by 4-fold) the expected increase observed with Mg2+ alone. Biochemical analyses of isolated S-FBP(PCM) revealed similarities to hydrophobic FBPs with respect to the ligand-binding domain and epitopes but differed in detergent-binding characteristics; furthermore, amino acid and carbohydrate analysis revealed a lower Mr = 25,500 with 12% carbohydrate. Based on kinetic analysis of S-FBP(PCM) release from chorionic villi-associated hydrophobic FBPs as well as structural analysis of S-FBP(PCM), these data continue to support the hypothesis that (α) a significant amount of maternal and probably fetal serum hydrophilic FBPs originate from placental hydrophobic FBPs, and (b) the endogenous hydrophobic FBP-directed Mg2+-dependent placental protease plays a major role in their release.

AB - Full term placental chorionic villi cultured for 7 days in serum-free medium released hydrophilic folate-binding proteins (S-FBP(PCM)) into the conditioned medium; in contrast, hydrophobic FBPs were the only form recovered from chorionic villi. Kinetic studies revealed that (i) S-FBP(PCM) was maximally released by the 3rd day, and this was associated with a proportionate decrease in hydrophobic FBPs; (ii) although cycloheximide inhibited de novo synthesis of [35S]methionine-labeled hydrophobic FBPs and S-FBP(PCM) by >90%, unlabeled net S-FBP(PCM) release was only inhibited by 50%; (iii) EDTA markedly inhibited release of S-FBP(PCM) which was accompanied by a proportionate increase in hydrophobic FBPs; (iv) EDTA effects were completely reversed by 5-fold molar excesses of Mg2+ which led to a 50-fold greater release of S-FBP(PCM) compared to EDTA alone; (v) whereas Mg2+ alone stimulated S-FBP(PCM) release 4-fold greater than basal conditions, addition of cycloheximide to Mg2+ suppressed (by 4-fold) the expected increase observed with Mg2+ alone. Biochemical analyses of isolated S-FBP(PCM) revealed similarities to hydrophobic FBPs with respect to the ligand-binding domain and epitopes but differed in detergent-binding characteristics; furthermore, amino acid and carbohydrate analysis revealed a lower Mr = 25,500 with 12% carbohydrate. Based on kinetic analysis of S-FBP(PCM) release from chorionic villi-associated hydrophobic FBPs as well as structural analysis of S-FBP(PCM), these data continue to support the hypothesis that (α) a significant amount of maternal and probably fetal serum hydrophilic FBPs originate from placental hydrophobic FBPs, and (b) the endogenous hydrophobic FBP-directed Mg2+-dependent placental protease plays a major role in their release.

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