Kinetic analysis, isolation, and characterization of hydrophilic folate-binding proteins released from chorionic villi cultured under serum-free conditions.
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 greater than 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 (a) a significant amount of maternal and probably fetal serum hydrophilic FBPs originate from placental hydrophobic FBPs, and (b) the endogenous hydrophobic FBP-directed Mg(2+)-dependent placental protease plays a major role in their release.
Verma
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RS
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Antony
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AC
D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D002824:Chorionic Villi / Q000737:chemistry* / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D011233:Precipitin Tests; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D003513:Cycloheximide; D008715:Methionine