Alanine transport across the human placental brush border membrane and the role of SH groups in carrier function.
We have determined the kinetic characteristics of alanine transport into brush border membrane vesicles (BBMV) of human full term placenta and identified functional groups of the carrier proteins that are important for transport function. Alanine influx into BBMV was found to be mediated by two transport systems with different kinetic features and distinct substrate specificities. An uphill operating electrogenic Na(+)-dependent cotransport system could be kinetically separated from a Na(+)-independent facilitated diffusion system. The Na(+)-dependent transporter mediates Na(+)-alanine cotransport with a 1:1 flux coupling ratio (Hill coefficient 1.13 +/- 0.12) and a Km for alanine of 0.45 +/- 0.06 mmol/l. Half-maximal stimulation of Na(+)-dependent alanine influx was observed at a Na+ concentration (NaCl) of 51.4 +/- 1.3 mmol/l. A variety of group specific reagents were used to identify functional groups in the transport proteins. Only compounds reacting with SH-residues (NEM, DTNB, PCMBS) or NH2-groups (PITC) were found to affect Na+ dependent and Na+ independent alanine transport. The EC50 value for inhibition of alanine influx by PCMBS was 450 +/- 48 mumol/l. Chemical modifications of SH-groups by PCMBS caused a significant reduction (p < 0.005) in the Vmax for Na(+)-dependent alanine influx from 0.57 +/- 0.06 to 0.16 +/- 0.05 nmol.mg protein-1.10s-1 without affecting significantly the Km value. Inhibition by PCMBS was reversed by treatment of BBMV with DTT. When the substrate binding site of the transporter was protected by alanine or leucine, PCMBS still, blocked transport function, indicating that the crucial SH groups are not located within the substrate binding site of the transport proteins.
Scholl
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A
AG Biochemie der Ernährung, Institut für Ernährungswissenschaft, Justus-Liebig-Universität Giessen.
Gent
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Daniel
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H
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000409:Alanine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000494:pharmacology; D008431:Maternal-Fetal Exchange; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D012965:Sodium Chloride / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D018698:Glutamic Acid; D012965:Sodium Chloride; D002731:4-Chloromercuribenzenesulfonate; D007930:Leucine; D000409:Alanine