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 in to brush border membrane vesicles (BBMV) of human full term placenta a nd identified functional groups of the carrier proteins that are impor tant 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 transpor ter mediates Na+-alanine cotransport with a 1:1 flux coupling ratio (H ill coefficient 1.13 +/- 0.12) and a Km for alanine of 0.45 +/- 0.06 m mol/l. Halfmaximal stimulation of Na+-dependent alanine influx was obs erved 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 EC(50) value for inhibition of alanine influx by PCMBS was 450 +/- 48 mu mol/l. Chemical modifica tions 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 significa ntly the Km value. Inhibition by PCMBS was reversed by treatment of BB MV with DTT. When the substrate binding site of the transporter was pr otected by alanine or leucine, PCMBS still blocked transport function, indicating that the crucial SH groups are not located within the subs trate binding site of the transport proteins.