Sulphydryl modification inhibits taurine transport in human placental brush border membranes

Placenta (1996), 17, 329-336

Sulphydryl Brush

Modification

Border

R. Dumaswalaa a Division

Taurine

Transport

in Human

Placental

Membranes

and T. L. Brownb

of Pediatric

b Developmental Paper accepted

Inhibits

Gastroenterology

Biology 4 March

Program, 1996

and Nutrition,

University

Children’s

of Cincinnati,

Hospital

Cincinnati,

Research

OH 45229,

Foundation,

Cincinnati,

OH 45229,

USA

USA

Taurine, 2-aminoethanesulphonic acid, is a -amino acid required for mammalian development. Although the human fetus accumulates taurine in many tissues, it has limited capacity for synthesis. The majority of fetal taurine is derived from the mother via placental transfer. The objective of this study was to analyse the functional groups involved in the taurine transport system of human placental brush-border membranes. Sulphydryl modifying reagents N-ethylmaleimide (NEM) and pyridyldithioethylamine (PDA) caused a dose-dependent inhibition of taurine uptake by brush-border membrane vesicles. Inhibition by PDA was reversible upon reduction by dithiothreitol but not by glutathione indicating that sulphydryl group(s) are located within the bilayer. Preincubation of brush-border membranes with taurine but not with taurocholate, before exposure to NEM, protected taurine transport function. Labelling studies using NEM and chemical cross-linking indicated that a 37.5 kDa protein was protected. These results demonstrate that sulphydryls located within the membrane bilayer are important for taurine transport in human placental brush-border membranes and suggest that a 37.5 kDa protein may be associated with Na+-dependent regulation of the taurine transporter. 0 1996 W. B. Saunders Company Ltd Placenta (1996), 17, 329-336

INTRODUCTION The human fetus accumulates taurine (2-aminoethanesulphonic acid), a ubiquitous sulphoamino acid, in many tissues during development (Chesney, 1985; Wright et al., 1986; Kendler, 1989). During this period of rapid growth, the fetus has only a limited capacity to synthesize taurine, and is entirely dependent upon exogenously supplied taurine (Sturman, Rassin and Gaull, 1977). Therefore, placental transport of taurine is critical for normal growth and development of the fetus. Maternal taurine deficiency leads to growth retardation in fetal kittens, rat pups and monkeys (Hayes, Stephan and Sturman, 1980; Huxtable, 1982; Sturman et al., 1986). Thus, in a developmental setting of increased demand and decreased synthetic capacity, maternal taurine is an essential fetal nutrient. Taurine transport across brush-border membranes (BBM) of syncytiotrophoblasts isolated from human term placenta has been well characterized (Miyamoto et al., 1988; Hibbard et al., 1990; Karl and Fischer, 1990; Moyer, Insler and Dumaswala, 1992). Human placental BBM actively transport taurine by the p-amino acid carrier, similar to other tissues (Athee, Baillin and Paasonen, 1974; Rosen, Tenenhouse and Striver, 1979; Meiners et al., 1980; Schmidt and Berson, 1980; Franconi et al., 1981; Ohkuma et al., 1984; Chesney et al., 1985; a To whom correspondence

should be addressed

0143U!OO4/96/050329+08 $12.00/O

Lambert, 1985; Bucuvalas, Goodrich and Suchy, 1987; Barnard et al., 1988; Moyer et al., 1988; Lewis, Cohen and Smith, 1990). The p-amino acid carrier system is sodium and chloride-dependent, saturable, and inhibitable by p-amino acids (Miyamoto et al., 1988; Hibbard et al., 1990; Karl and Fischer, 1990; Moyer, Insler and Dumaswala, 1992). Recently, a human placental taurine transporter has been described, and has a predicted molecular weight of 70 kDa (Ramamoorthy et al., 1993a,b; Ramamoorthy et al., 1994). Taurine has been implicated in numerous physiological functions, including bile acid metabolism (Huxtable, 1992), osmoregulation of cell volume (Kendler, 1989), and intracellular Ca2+ homeostasis (Huxtable, 1990; Huxtable, 1992). Tyrosine residues have been shown to be essential for placental taurine transport (Kulanthaivel et al., 1989), however, the involvement of other functional groups and protein(s), which regulate placental taurine transport, remains to be determined. In order to better understand taurine transport and proteins involved in its regulation, we have analysed functional groups important in taurine binding and translocation, and subsequently examined taurine interactions with endogenous BBM proteins. Our results demonstrate sulphydryl groups located within the membrane bilayer are important in the function of the taurine transporter. The results also suggest that sulphydryl modification of a 37.5 kDa protein may be associated with regulation

of the taurine 0

transporter.

1996 W. B. Saunders

Company

Ltd

330

Placenta

(1996),

Vol. 17

”

DIDS

PITC

Phenyl glyoxal Inhibitors

NEM

PDA

DTT

(0.5 mM)

Figure 1 Sensitivity of the taurine transporter and sulphydryl group modifying reagents. BBM vesicles were treated with 0.5 mM DIDs, PITC, phenyl glyoxal, NEM, PDA or DTT for 30 min at room temperature. The membranes were washed to remove excess reagent. Initial rate (10 set) of Na+-dependent taurine (1 PM) uptake was determined and compared with that of untreated controls. Data are presented as per cent of control values and are the means of i s.d. of triplicate determinations from three different preparations (“P< 0.05 versus control).

MATERIALS

AND

METHODS

Materials

[3H]-Taurine (Sp.Act.20.1 Ci/mmol) was obtained from DuPont New England Nuclear (USA). Unlabelled taurine, N-ethylymaleimide (NEM), phenylglyoxal, phenylisocyanate (PITC), 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS), dithiothreitol (DTT) and glutathione were purchased from Sigma, USA. All other chemicals were of the highest grade commercially available. Pyridyldithioethylamine (PDA) was synthesized by the procedure described by Johnson and Chenoweth (Johnson and Chenoweth, 1985).

Preparation

of BBM

vesicles

Normal human term placentae were obtained within 30 min of delivery and processed immediately. Syncytiotrophoblast BBM vesicles were prepared according to the procedure described by Karl, Teichberg and Fischer (Karl, Teichberg and Fischer, 1991). Vesicle preparations for transport studies were stored at 4°C and used within 2 days. Protein was quantitated as described by Bradford (Bradford, 1976). The purity of BBMs was monitored by assaying alkaline phosphatase and Na+-K+ ATPase, marker enzymes for BBM and basal membranes of the human placenta, respectively (Booth, Olaniyan and Vanderpuye, 1980).

Transport

and

inhibition

studies

All uptake measurements were performed at 30°C using a rapid-quench Millipore filtration technique described by

Dumaswala et al. (Dumaswala et al., 1993). Uptakes were initiated by mixing 20 ~1 of membrane suspension (60-80 pg protein) with 80 ~1 of incubation buffer containing labelled taurine (see figure and table legends for specific composition). Timed uptakes were terminated by addition of 3 ml of ice-cold stop solution (10 mM HEPES-Tris, pH 7.4, 100 mM NaCl or KC1 and 100 mM mannitol). The mixture was rapidly filtered through a nitrocellulose Millipore filter (HA, 0.45 pm) that had been presaturated with taurine to reduce nonspecific binding. The test tube was rinsed with 3 ml stop solution and the contents filtered. The filter was washed twice with 3 ml stop solution, dissolved in 4.0 ml Ready-Protein (Beckman Instruments, USA) and counted in Beckman LS-3801 liquid scintillation counter. A blank to correct for nonspecific binding of taurine to the filter was determined in each experiment by adding 3.0 ml of ice-cold stop solution to the vesicles before the addition of [3H]-taurine. All transport measurements were performed in quadruplicate, and each experiment repeated with at least three different membrane preparations. For inhibition studies, BBM were incubated with the respective group specific reagents, as indicated in the figure legends, for 30 min at room temperature. The membranes were washed as described above and analysed for their ability to transport taurine as compared with untreated controls.

Substrate

protection

and

labelling

with

[3H]-NEM

BBM were incubated with or without substrate (taurine or taurocholate) for 30 min at room temperature followed by exposure to unlabelled NEM. NEM binds to sulphydryl groups irreversibly and is not removed by washing the BBM. After exposure to unlabelled NEM, the BBM were

Dumaswala

and Brown:

Sulphydryls

Are Important

in Placental

Taurine

331

Transport

(a)

0.1

0.25

0.5 NEM

(b)

1.0

2

2.0

(mM)

15 ” 60

120 (min)

Figure 3. Effect of NEM treatment on sodium-dependent taurine uptake by BBM. BBM were incubated with (A, 0) or without (a, 0) 0.5 mM NEM at room temperature for 30 min. The membranes were washed to remove excess reagent and vesiculated. Timed uptake of taurine was measured in the presence of an inwardly directed 100 mM Na+ (a, A) or Kf (0, 0) chloride gradient. Results are given as means of & s.d. of triplicate determinations from three membrane preparations.

I-

T /-

0.05

0.1 PDA

0.25

0.5

1.0

(mM)

Figure 2. Concentration dependence of inhibition of taurine influx. The BBM vesicles were incubated with indicated concentrations of NEM (a) and PDA (b) at room temperature for 30 min. The membranes were washed to remove excess inhibitor and suspended in 10 mM HEPES/ KOH (pH 7.4) containing 300 mM mannitol as described in Methods. Initial rate (10 xc) of sodium-dependent taurine (1 FM) was measured. Results are given as * s.d. of 12 determinations from three different experiments.

subsequently washed to remove excess NEM, and all of the substrate (taurine), and then used to measure taurine/ taurocholate influx. BBM were protected with substrate (0.1-0.5 rnM taurine), incubated with unlabelled NEM, and washed as described as above. The substrate (taurine) protected and washed BBM were then exposed to [3H]-NEM. Only sulphydryl groups protected by the substrate (taurine) will have free sulphydryls capable of reacting with [3H]-NEM. After washing the labelled BBM, the radiolabelled membranes were solubilized and analysed on a 10 per cent sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) as described by Laemmli (Laemmli, 1970). The gels were stained with 0.1 per cent Coomassie blue in 10 per cent glacial acetic acid and 0.5 per cent methanol, dried and developed by autoradiography. Autoradiograms were scanned using a densitometer.

Cross-linking

10

Time

T

0.025

5

C3H]-taurine

to

placental

BBM

For cross-linking studies, BBM were incubated [3H]-taurine (20 PM) or [3H]-taurocholate (20 PM)

with and

N-hydroxysulphosuccinimide (Pierce), a lipophilic, membrane permeable cross-linker, for 30 min at room temperature. Coupling was initiated by addition of 1-cyclohexyl-3-(2morpholinoethyl) carbodiimide metho-p-toulenesulphonate (EDC) (Staros et al., 1986). The reaction was terminated between 2-30 min by the addition of an equal volume of 0.5 mM Tris (pH 7.4). The proteins were analysed by 10 per cent SDS-PAGE and the gels were stained, destained, enhanced, dried and autoradiographed.

Statistical

analysis

Uptake measurements were routinely performed in quadruplicate and the variation among the replicate values was always less than f 5 per cent of the mean value. Each experiment was repeated with at least three different membrane preparations. Results are given as the mean ZIZse. Statistical differences were determined by Student’s t-test; P values less than 0.05 were considered significant.

RESULTS

Group-specific reagents are capable of modifying reactive groups on amino acids and affecting subsequent transport activity. Therefore, we tested the sensitivity of the human placental BBM taurine transporter to amino and sulphydrylgroup modifying reagents (Figure 1). The presence of critical sulphydryl-groups in the taurine transporter was suggested by sulphydryl modifiers NEM and PDA, both of which inhibited sodium-dependent taurine (1 PM) transport by 60-80 per cent. DIDS, which preferentially modifies the epsilon amino group of lysine, inhibited taurine transport in BBM but to a lesser extent (40 per cent). In contrast, phenyl glyoxal, a specific

Placenta

332

(1996),

Vol. 17

Figure 4. Reversibility of PDA inhibition by DTT and glutathione. Untreated controls and BBM vesicles were pretreated with 0.1 rn~ PDA for 30 min at room temperature. After washing and centrifugation, the BBM vesicles were incubated 10 min with DTT or glutathione at 37°C and the initial rate (10 set) of sodium-dependent taurine (1 PM) uptake was subsequently measured in the presence of either 5 or 10 mM DTT or glutathione. Results are expressed as per cent of control and represent means & s.d. of 12 determinations from three different membrane preparations (“P
Table

1. Effect of substrate

protection

in the

presence of NEM on taurine/taurocholate Uptakes (pmol/mg

Substrate

Inhibitor

TR (0)

NEM NEM NEM NEM

TR (0) TR TC

(0.25 (0.25

mM) ITIM)

_

(0) (0.5 ITIM) (0.5 ItIM) (0.5 mM)

transport

protein)

Taurine

Taurocholate

6.90 zk 0.35 1.80 zt 0.20 4.70 zt 0.23 1.68 * 0.12

4.51 2.63 2.70 3.90

* 0.28 zt 0.11

* 0.19 It 0.27

The BBM were incubated with or without substrate [0.25 mM taurine (TR) or taurocholate (TC)] for 30 min at room temperature before exposure to NEM. The membranes were washed to remove substrate and excess NEM and resuspended in buffer containing 10 mM HEPES (pH 7.4) and 300 mM mannitol. Initial rate (10 set) of r3H]-taurine (1 PM) and taurocholate (1 PM) transport was measured Values are the means f s.e. of four determinations of a representative experiment.

reagent for the guanidino group of arginine and PITC, specific for the terminal amino group, had no significant effect on taurine transport. To further analyse the sulphydryl groups that could regulate placental taurine transport, dose-response measurements were performed with NEM and PDA. NEM and PDA inhibited taurine (1 PM) transport by BBM vesicles in a dose-dependent manner, with IC,, values of 0.5 mM and 0.075 mM, respectively (Figure 2). Taurine transport in the presence of inwardly directed KC1 gradient, as well as equilibrium uptake values measured after 120 min of incubation remained unchanged, demonstrating that the integrity of the membranes was not affected by NEM treatment (Figure 3). PDA is a reversible thiol-disulphide exchange reagent (Connor and Schroit, 1988). Therefore, it is possible to inhibit taurine transport and yet reverse its effect following bond reduction. To determine whether important sulphydryl group(s) for taurine transport are located within the membrane bilayer, the effect of two sulphydryl-reducing agents, DTT and glutathione, on PDA

inhibition of taurine transport were examined (Figure 4). PDA inhibition was reversed by DTT but not by glutathione, suggesting that the sulphydryl groups modified by PDA, and critical for taurine transport, are likely to be located within the interior of the bilayer. Inhibition by NEM, an irreversible sulphydryl modifier, was not affected by either DTT or glutathione. Modification of biological membranes by sulphydrylreactive agents inhibits many transport systems, including Cl- transport (Scott et al., 1970; Solberg and Forte, 1971). To determine whether NEM-dependent inhibition of taurine transport was the result of direct carrier modification or a secondary effect of Cl - inhibition, we examined the ability of taurine to protect the p-amino acid carrier from NEM inactivation. The protective effect of taurocholate, a non-substrate, was also examined because taurocholate has amidated taurine moiety and its transport is also sensitive to NEM-dependent sulphydryl modification (Blumrich and Petzinger, 1990). Taurine transport activity of placental

Dumaswala 1

and Brown:

Sulphydryls

2

3

Are Important 4

5

in Placental

Taurine

Transport

pretreated with taurine (0.1-0.5 mM) followed by incubation with unlabelled NEM, as described in Methods. The BBM were washed to remove excess unlabelled NEM and all of the taurine, and subsequently allowed to react with [3H]-NEM. Because NEM binding to sulphydryl groups is irreversible, only free sulphydryls that were protected by taurine pretreatment will be accessible for [3H]-NEM labelling. Protection with increasing concentrations of taurine resulted in a doseresponsive increase in labelling of a 37.5 kDa protein (Figure 5, Table 2). The specificity of this protection was demonstrated by the fact that p-amino acids, taurine, hypotaurine and p-alanine but not a-amino acids, valine or leucine, could protect the 37.5 kDa protein against NEM modification (Figure 6). A protein of molecular weight 43 kDa was also labelled and was identified as actin as determined by cross-reactivity on a Western blot (data not shown). In order to confirm initial observations that a 37.5 kDa protein was important in taurine transport, a more direct approach, chemical cross-linking, was used to identify taurine binding proteins. [3H]-taurine was cross-linked to placental BBM. The amino group of taurine can react with carboxylic acids to form amide linkages (Skare, Schnoes and DeLuca, 1982). Similarly, the sulphonic acid group can be amidated to form sulphonamides (Andersen et al., 1983). Cross-linking of taurine to BBM was initiated by membrane permeable crosslinker NHS and catalysed by EDC. Autoradiographic analysis of the cross-linked BBM proteins indicated intense labelling of two proteins of 37.5 and 35 kDa (Figure 7). [3H]-taurocholate under similar conditions labelled BBM proteins of MW 70, 58 and 43 kDa (Figure 7). Cross-linking of taurine to the BBM resulted in loss of taurine transport activity compared with untreated membranes as well as those treated with NHS-EDC alone (Figure 8).

6

kDa 97.4

66.2

42.7

31 21

Figure 5. Labelling of BBM vesicles with [%-NEM. BBM vesicles were incubated with unlabelled NEM (0.5 mM) and increasing concentrations of taurine for substrate protection. The membranes were washed to remove excess unlabelled NEM and the substrate protected BBM vesicles were exposed to [3H]-NEM for 30 min at room temperature. The radiolabelled proteins were analysed by 10 per cent sodium dodecyl sulphatepolyacrylamide gel electrophoresis and fluorography. BBM treated with [H-NEM (lane 1). The BBM vesicles were protected with taurine at 0.1 mM (lane 2), 0.2 mM (lane 3), 0.3 rnM (lane 4), 0.4 mM (lane 5) and 0.5 tTIM (lane 6) before exposure with unlabelled NEM. The membranes were then washed and exposed to C3H]-NEM for labelling. The 37.5 kDa protein is indicated by the solid arrow and actin is identified by the solid arrowhead.

BBM vesicles could be protected by preincubation of the membranes with taurine, but not taurocholate, before exposure to NEM (Table 1). These results suggest that NEM-dependent inhibition of taurine transport is due to modification of the carrier sulphydryl group(s) at or near the taurine binding site. To identify the sulphydryl-modified proteins involved in the regulation of taurine transport, placental BBM were

Table

2. Concentration-dependent

Taurine concentration (ml@

Actin 37.5 kDa Densitometric experiments represented

protein

protective

effect

333

DISCUSSION The results of the present study demonstrate that the taurine transport in human placental BBM vesicles is inhibited by sulphydryl modifying reagents NEM and PDA. Inhibition by PDA, a thiol-disulphide exchange reagent, is reversed by the presence of membrane penetrating thiol, DTT, but not by nonpenetrating thiol glutathione (Connor and Schroit, 1988).

of taurine by NEM

on sulphydryl

modification

of 37.5 kDa

protein

0

0.1

0.2

0.3

0.4

0.5

100 15.8 =t 3.6

100 2.80 dc 0.6

100 3.00 f 0.8

100 5.20 r!c 1.0

100 8.70 + 1.5

100 11.3 Zt 2.1

scanning of [3H]-NEM labelling of BBM were repeated th ree times and the values in per cent * s.e.

vesicles. Mean densitometric scan of 37.5 kDa protein. The for the 37.5 kDa protein were normalized to actin and are

Placenta

334

1

2

3

4

5

1

6

2

3

4

5

(1996),

Vol. 17

6

kDa

kDa 66.2

70 63

42.7

43

31

37.5 35

21 11 Figure 6. Specificity of [%-r\TEM labelling. BBM vesicles were preincubated with no amino acids (lane 1) or with 0.1 mM of the following: alanine (lane Z), valine (lane 3), leucine (lane 4), taurine (lane 5) or hypotaurine (lane were 6) before the addition of unlabelled NEM (0.5 tTIM). The membranes subsequently washed to remove the substrate and unreacted reagent, the membranes were exposed to r3H]-NEM. The labelled proteins were resolved by 10 per cent sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by fluorography. The 37.5 kDa protein is identified by the solid arrow.

These results suggest that the sulphydryl group(s) critical for taurine transport are located within the interior of the membrane bilayer. In addition, the ability of the placental BBM to transport taurine into vesicles depends on the maintenance of these sulphydryls in a reduced state. Recently, interest has focused on the role of sulphydryl groups in membrane carrier proteins, and their functional importance in the regulation of the substrate transport across biological membranes. Numerous transport systems have been analysed kinetically and have been shown to be sensitive to sulphydryl modification (Schaeffer, Preston and Curran, 1973; Hayes and McGivan, 1983; Vitanen et al., 1985; Chiles and Kilberg, 1986; Chiles, Dudeck-Collart and Kilberg, 1988; Ibarra, Ripoche and Bourguet, 1989; Blumrich and Petzinger, 1990). However, interpretation of kinetic data based on sulphydryl modification may be misleading in that inhibition or increased transport may be assumed to be a direct interaction with the transporter and may not account for protein complex formation and regulation of the transport process. Although few investigations have utilized thiol-group specific reagents to identify proteins involved in the regulation of transport systems, confirmation of direct or indirect sulphydryl interactions with

Figure 7. Cross-linking of [%-taurine to BBM. BBM were incubated with [3H]-taurine for 90 min at room temperature. The cross-linking reaction was initiated by addition of NHS and carbodiimide. The timed reaction (10 sec15 min) was stopped by addition of equal volume of 0.5 M Tris and 4X Laemmli buffer. The labelled proteins were analysed by 10 per cent sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by fluorography. Lane l-BBM cross-linked with [%-taurocholate for 1 min. Lanes 2-6 represent BBM cross-linked with [3H]-taurine for 0.5, 1, 5, 10, and 15 min, respectively.

a proposed transporter is important for a complete understanding of the regulatory mechanisms involved. NEM has been used to identify proteins involved in alanine transport in rat liver membrane (Hayes and McGivan, 1983) and D-glucose transport in kidney BBM (Poiree, Mengual and Sudaka, 1979). Because NEM is a nonspecific inhibitor, identification of a carrier protein required the demonstration of substrate protection from binding NEM in the presence of the transport substrate. In the present studies, a thiol-sensitive, taurine-binding protein was identified by labelling the substrate protectable sulphydryl group(s) with [3H]-NEM. The intensity of a labelled 37.5 kDa protein is enhanced when the BBM are protected with increasing concentrations of taurine These results suggest that taurine protects (0.1-0.5 mM). sulphydryl group(s) of the 37.5 kDa protein from NEM modification, and thus, alleviates inactivation of sodiumdependent taurine transport. Protection of taurine transport observed by preincubation of the BBM vesiclesbefore addition

Dumaswala

and Brown:

0.25 0.5

Sulphydryls

Are Important

in Placental

2.0

1.0

Time

5.0

Taurine

”

180

(min)

Figure 8. Effect of cross-linking on taurine transport BBM were cross-linked with ( /?, ) or without ( D ) membranes were washed to remove excess reagents timed uptake of [%-taurine was determined and untreated controls ( 0 ). Data are means f s.d. of three different experiments.

335

Transport

by BBM vesicles. The taurine for 20 sec. The and sodium-dependent compared with that of 16 determinations from

of NEM, with taurine but not with taurocholate, indicates that inhibition of taurine transport was due to a binding of NEM to the sulphydryl groups, and suggests that these sulphydryl groups are at or near the taurine binding site of the carrier protein. To determine if the 37.5 kDa protein was capable of

binding taurine, we used an alternate approach to cross-linking of taurine to the BBM. A 37.5 kDa protein was heavily labelled, confirming earlier results with NEM that a 37.5 kDa protein binds taurine. Taken together, the results suggest that sulphydryl groups are important for taurine transport, and that a 37.5 kDa protein may play a regulatory role in human placental taurine transport. An association of a 37.5 kDa protein with taurine transport is suggested because taurine binds to the 37.5 kDa protein and the chemical modification of the sulphydryl group(s) of the 37.5 3kDa protein inhibits taurine transport. Surprisingly, no labelling of the 70 kDa taurine transporter was detected (Ramamoorthy et al., 1994), therefore, a regulatory role of 37.5 kDa in taurine transport may be possible. It is not clear why the placental taurine transporter was not labelled in taurine cross-linking experiments. It is possible, however, that the taurine transporter is not directly accessible to taurine or that the number of transporters present is below detectability of [3H]-cross-linking. Alternatively, the activation of the transporter may require taurine binding to other proteins as an initial step. Therefore, it is possible that the 37.5 kDa protein may be part of a taurinetransport complex. Further studies are needed to determine the role of the 37.5 kDa protein in placental taurine transport.

ACKNOWLEDGEMENTS This work was supported in part by a Bristol-Myers p&natal research award (R.D.), National Institutes of Health Grant HL 27333 (Dr J. K. Harmony) National Institutes of Health predoctoral training grant HL07527 (T.L.B.). We are grateful to Drs J. A. K. Harmony, J. R. Dedman, M. Kaetzel, J. E. Heubi W. F. Balistreri for helpful discussions on this manuscript. Special thanks to Dr J. Lessard for providing the actin antibody.

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and and

inactivation by sulphydryl-modifying reagents. Joburnal of Cellular Physiology, 129, 321-328. Chiles, T. C., Dudeck-Collart, K. L. & Kilberg, M. S. (1988) Inactivation of amino acid transport in rat hepatocytes and hepatoma cells by PCMBS. American Journal of Physiology, 255, C340-C345. Connor, J. & Schroit, A. J. (1988) Transbilayer movement of phosphatidylserine in erythrocytes: inhibition of transport and preferential labelling of a 3100-dalton protein by sulphydryl reactive reagents. Biochemistry, 27, 848-851. Dumaswala, R., Setchell, K. D. R., Moyer, M. S. & Suchy, F. J. (1993) An anion exchanger mediates bile acid transport across the placental microvillous membrane. American Journal of Physiology, 264, G1016G1023. Franconi, F., Martini, F., Manghi, N., Galli, A., Bennardini, F. & Giotti, A. (1981) Uptake of [j]H-taurine into myocardial membranes. Biochemical Pharmacology, 30, 77-80. Hayes, K. C., Stephan, 2. F. 8i Sturman, J. A. (1980) Growth depression in taurine-depleted infant monkeys. Journal of Nutrition, 110, 2058-2064. Hayes, M. R. & McGivan, J. D. (1983) Comparison of the effects of certain thiol reagents on alanine transport in plasma membrane vesicles from rat liver and their use in identifying the alanine carrier. Biochemical Journal, 214, 489495. Hibbard,

J. U.,

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Whitington,

P. F. and

Moawad,

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