Introduction

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cDDP is a very potent drug effective against a variety of solid tumors, but its clinical use is limited by its acute nephrotoxic potential. cDDP has been shown to impair reabsorption by renal epithelia, leading to polyuria, magnesium and sodium wasting and glucosuria (Fillastre and Raguenez-Viotte, 1989; Safirstein et al., 1987; Goldstein et al., 1981). In rats treated with a single low dose of cDDP, glucosuria appeared at an early stage, before the elevation of blood urea nitrogen levels (Goldstein et al., 1981). Glucosuria may result from an impairment of the low-affinity Na⁺/glucose cotransport system, which is responsible for the reabsorption of the bulk of glucose by cells in the early proximal convoluted tubule (Elsas and Longo, 1995). Moreover, cDDP inhibits the low-affinity Na⁺/glucose cotransporter in renal proximal tubular cells in primary culture (Courjault-Gautier et al., 1995) and in BBM vesicles prepared from the renal cortex of animals exposed to cDDP in vivo (Halabe et al., 1991; Yanase et al., 1992). The cDDP-induced inhibition of Na⁺-dependent glucose uptake may result from a direct interaction between cDDP and the cotransport protein, because this inhibition has been observed in BBM vesicles exposed to cDDP in vitro (Courjault-Gautier et al., 1995).

Several experimental findings suggest that cDDP-induced inhibition of the low-affinity Na⁺/glucose cotransport system could result from covalent platinum binding to SH groups of the transporter. The Na⁺/glucose cotransport protein in renal cortex BBM vesicles possesses SH groups that are essential for its activity (Lo and Silverman, 1994). HgCl₂, a specific SH reagent, inhibits the Na⁺-dependent glucose uptake in BBM vesicles from cortex of rat kidney (Kintziger and Kuntziger, 1986). Moreover, cDDP inhibits the activity of several proteins with SH groups essential for their activity, such as the Na⁺/phosphate cotransporter (Courjault-Gautier et al., 1995; Yanase et al., 1992), Na⁺-K⁺-ATPase (Courjault-Gautier et al., 1994; Uozomi and Litterst, 1985) and -glutamyltranspeptidase (Dedon and Borch, 1987). In the rat renal proximal tubule, cDDP induces a decrease in protein-bound thiols which, like glucosuria, occurs before the elevation of blood urea nitrogen levels (Mistry et al., 1991). Platinum (II) shows high affinity for sulfur-containing species such as protein-bound SH groups (Howe-Grant and Lippard, 1980; Melius and Friedman, 1977). Moreover, cDDP forms aquated or hydroxylated derivatives that are highly reactive for biological nucleophiles (fig. 1). cDDP transformation through aquation reactions (chemical exchanges between cDDP chloride ligands and water) are promoted by low chloride ion concentrations (intracellular-like concentrations), whereas high chloride ion concentrations (extracellular-like concentrations) stabilize the chemical structure of cDDP in the dichloro form (Howe-Grant and Lippard, 1980; Segal and Le Pecq, 1985). The presence of these reactive hydrated derivatives in chloride-free medium could explain why cDDP-induced inhibition of -glutamyltranspeptidase and leucine aminopeptidase is more potent in the absence than in the presence of chloride ions (Dedon and Borch, 1987).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Aquation reaction of cDDP.

Based on the affinity of platinum for sulfur-containing species, several nucleophiles have been studied as chemoprotective agents against cDDP nephrotoxicity (Pinzani et al., 1994; Treskes and van der Vijgh, 1993). Among these chemoprotectors, DDTC has been shown both to remove platinum bound to some biological nucleophiles, resulting in the formation of Pt(DDTC)₂ (Bodenner et al., 1986; Dedon and Borch, 1987; Lempers and Reedijk, 1990), and to restore, at least in part, several enzyme activities inhibited by cDDP. This restoration may result from platinum removal from methionine or cysteine residues (Bodenner et al., 1986).

This study investigated, with use of BBM vesicles from the outer cortex of rabbit kidney, the direct interaction of cDDP with the low-affinity Na⁺/glucose cotransporter, the effect of cDDP on protein-bound thiols and platinum binding to BBM vesicles in the presence and absence of low and high chloride ion concentrations. DDTC was used to clarify the role of platinum binding to protein-bound thiols in the cDDP-induced inhibition of the Na⁺/glucose cotransporter.

	Materials and Methods

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Drugs and chemicals. cDDP, DDTC, 5,5-dithiobis-(2-nitrobenzoic acid) and MGP were from Sigma Chemical Co. (St. Louis, MO). [U-¹⁴C]MGP (293 mCi/mmol) and diethyl-[¹⁴C]dithiocarbamic acid (25 mCi/mmol) were from Amersham (Amersham, UK). Nitrocellulose filters (pore size, 0.65 µm) were from Sartorius (Palaiseau, France). Ultima Gold scintillation fluid was from Packard Instrument Co. (Meriden, CT). All other reagents were of analytical grade.

Mass spectrometry analysis. cDDP was dissolved at a concentration of 3 mM in buffered solution consisting of 150 mM NaCl and 20 mM HEPES, pH 7.4 (150 mM NaCl solution), 30 mM NaCl, 240 mM mannitol and 20 mM HEPES, pH 7.4 (30 mM NaCl solution) or 300 mM mannitol and 20 mM HEPES, pH 7.4 (NaCl-free solution). The mixtures were stirred at room temperature for approximately 8 hr to reach equilibrium among the hydrated complexes of cDDP. Before analysis, cDDP solutions were diluted in methanol at a final concentration of 0.75 mM. Samples were introduced into the ion source via a silica capillary with a 100-µm internal diameter at a flow rate of 10 µl/min. The voltage at the tip of the capillary, used at atmospheric pressure, was 5000 V. Nitrogen (flow rate, 0.6-1.2 l/min) was used as the nebulizer gas along the capillary. Calibration was done with polypropyleneglycol to obtain one-mass-unit resolution in the mass range scanned (205-360 amu, in 2 msec/0.1). Electrospray mass spectra were recorded on a Sciex API-III spectrometer (2400 amu mass range).

Animals. All animal care and handling procedures were in accordance with the requirements of the American Association for the Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. New Zealand White rabbits weighing 1.5 to 2 kg were supplied by Dombes breeding laboratories (Chatillon sur Chalaronne, France); they were given a standard pelleted diet, and filtered tap water was provided ad libitum.

Preparation of BBM vesicles. BBM vesicles were prepared from the outer cortex of kidneys by the MgCl₂ precipitation technique (Booth and Kenny, 1974). Rabbits were sacrificed by intravenous injection of pentobarbital, and the kidneys were removed and placed in ice-cold phosphate-buffered saline. The cortical tissue was dissected free and homogenized in a buffered solution consisting of 50 mM mannitol, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM Tris-(hydroxymethyl)-aminomethane-HCl, pH 7.0 (buffer-to-tissue ratio of 30:1, vol/wt), at high speed in a Waring blendor for 1 min. After a centrifugation step at 200 × g for 2 min to eliminate large tissue debris, the homogenate was submitted to magnesium aggregation as described previously (Courjault-Gautier et al., 1995). The final pellet, containing enriched BBM vesicles, was suspended in the appropriate buffered solution (see "cDDP Treatment").

cDDP treatment. In all experiments cDDP was dissolved in buffered solution, similar to that used for suspension of BBM vesicles, at a concentration of 3 mM approximately 8 hr before use.

Na⁺-dependent uptake of MGP. Uptake of MGP was measured at 37°C by the semiautomatic procedure reported by Kessler et al. (1978). Uptake was initiated by automatic mixing of 5 µl of BBM vesicles with 100 µl of incubation medium containing 50 mM NaCl, 50 mM KCl, 100 mM glycerol, 0.75 µM valinomycin, 10 mM HEPES, pH 7.4, supplemented with [U-¹⁴C]MGP (8 µCi/ml) and appropriate concentrations of MGP. The incubation was stopped by automatic addition of 3.5 ml of stop solution containing 50 mM KCl, 200 mM glycerol, 10 mM HEPES, pH 7.4, kept at 4°C. The resulting mixture was poured onto prewetted 0.65-µm pore size nitrocellulose filters and washed twice with 4.5 ml of ice-cold stop solution by filtration. Uptake was also measured in sodium-free buffered solution containing 50 mM KCl, 200 mM glycerol, 0.75 µM valinomycin, 10 mM HEPES, pH 7.4. Filter membrane radioactivity was counted by liquid scintillation in 4 ml of scintillation fluid. Na⁺-dependent uptake of MGP was calculated by subtracting uptake in the absence of sodium from that in the presence of sodium after correction for nonspecific trapping.

Protein sulfhydryl content measurements. The protein SH content in BBM vesicles was determined in a final volume of 1 ml containing 1.8% sodium dodecyl sulfate, 0.6 mM 5,5-dithio-bis-2-nitrobenzoic acid and 100 mM Tris-(hydroxymethyl)-aminomethane, pH 7.4. The protein SH content was assayed by measuring the 2-nitrothiobenzoic acid formed from 5,5-dithio-bis-2-nitrobenzoic acid at 412 nm (Klip et al., 1979). An aliquot of each sample was assayed for protein content with bovine serum albumin as standard (Smith et al., 1985).

Determination of platinum content. Platinum concentrations were measured by flameless atomic absorption spectrophotometry with a Perkin-Elmer 5100 atomic absorption photometer equipped with a Zeeman effect correction system. Standard operating conditions consisted of a 60-sec dry cycle from 80 to 150°C, a 50-sec char cycle from 150 to 1200°C and a 5-sec atomization cycle at 2500°C. Samples were diluted with concentrated nitric acid. The platinum content was determined with respect to a calibration curve constructed from standard additions of platinum to BBM vesicle suspension. An aliquot of each sample was assayed for protein content with bovine serum albumin as standard (Smith et al., 1985).

Determination of DDTC bound to BBM vesicles. BBM vesicles were incubated for 4 hr at 37°C with cDDP (0-1 mM) in 150 mM KCl or KCl-free solution. The incubation was stopped by centrifugation at 30,000 × g for 20 min; BBM vesicles were then washed once by suspending the pellet in the incubation buffered solution, followed by centrifugation at 30,000 × g for 20 min. The final pellet was suspended at a concentration of 1.8 mg protein/ml in buffered solution supplemented with 2 mM DDTC and diethyl[¹⁴C]dithiocarbamic acid (0.5 µCi/ml). Incubation (37°C, 1 hr) was stopped by adding 5 ml of ice-cold phosphate-buffered saline. The resulting mixture was poured onto prewetted 0.65-µm pore size nitrocellulose filters and washed with 15 ml of ice-cold phosphate-buffered saline by filtration. Filter membrane radioactivity was counted by liquid scintillation in 8 ml of scintillation fluid. The radioactive counts for each sample, corrected for nonspecific trapping, were normalized with respect to protein content (Smith et al., 1985).

Data analysis. Data are expressed as means ± S.E.M. The experiments were performed in duplicate or triplicate with use of at least three separate preparations of BBM vesicles. The cDDP concentrations required to inhibit Na⁺-dependent MGP uptake by 50% (IC₅₀) were calculated from the linear regression between the cDDP concentration and the inhibitory effect of cDDP by a Log-Logit model. Statistical comparisons between IC₅₀ values, decreases in protein-bound SH content or platinum binding values were made by use of Student's two-tailed unpaired t test. K_m and V_max values were analyzed by use of Student's paired t test. A P value of 0.05 or less was considered statistically significant.

	Results

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Effect of chloride ion concentration on the formation of cDDP-hydrated derivatives. The hydrated derivatives of cDDP were formed only when the chloride ion concentration was low, whereas cDDP remained in the dichloro form in the presence of high chloride ion concentrations (fig. 2). The major peak in the mass spectrum of cDDP in 150 mM NaCl solution started at m/z 321 (fig. 2A) and was identified as [Pt(NH₃)₂(Cl)₂+Na]⁺ (Ehrsson et al., 1995; Poon and Mistry, 1991). The main peaks in the mass spectrum of cDDP in 30 mM NaCl solution (fig. 2B), which started at m/z 321, m/z 263 and m/z 229, corresponded to [Pt(NH₃)₂(Cl)₂+Na]⁺, [Pt(NH₃)₂(Cl)]⁺ (Ehrsson et al., 1995) and [Pt(NH₃)₂+H]⁺, respectively. The prominent cluster ions of cDDP in NaCl-free solution (fig. 2C) corresponded to [Pt(NH₃)₂(Cl)]⁺ (m/z 263) and [Pt(NH₃)₂+H]⁺ (m/z 229), whereas the cDDP became a minor constituent.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of chloride ion concentration on cDDP aquation. cDDP (3 mM) was dissolved and stirred for 8 hr in 150 mM NaCl (A), 30 mM NaCl (B) or NaCl-free solution (C) at room temperature before analysis. Electrospray mass spectra were obtained in neutral loss of 17-amu mode.

Effect of cDDP on Na⁺-dependent uptake of MGP. The time course of Na⁺-dependent uptake of MGP was studied in control and cDDP-treated BBM vesicles. In BBM vesicles incubated in 150 mM KCl, 30 mM KCl and KCl-free solutions for 4 hr, Na⁺-dependent uptake of MGP (1 mM) was linear for up to 6 sec in control and 0.5 mM cDDP conditions. Na⁺-dependent uptake of MGP remained linear during a 6-sec period up to 15 mM MGP in BBM vesicles incubated with or without 0.5 mM cDDP in 150 mM KCl or KCl-free solution for 4 hr.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of cDDP on Na+-dependent MGP uptake by BBM vesicles. BBM vesicles were exposed for 4 hr to increasing concentrations of cDDP in 150 mM KCl (A), 30 mM KCl (B) or KCl-free (C) solution. The uptake of 1 mM MGP was measured at 37°C for 6 sec. Na+-dependent uptake was obtained by subtracting MGP uptake in the absence of sodium from that in the presence of sodium. The data represent the mean ± S.E.M. of three separate preparations of BBM vesicles.

Effect of cDDP on protein-bound SH groups and platinum binding to BBM vesicles. In control BBM vesicles, the protein SH content was approximately 83 µmol/g protein and remained constant during a 10-hr incubation period, whatever the chloride ion concentration. cDDP induced a time- and concentration-dependent decrease in the protein SH content in BBM vesicles (fig. 4). At 0.5 mM cDDP in 150 mM NaCl solution, the protein SH content in BBM vesicles represented 78, 68 and 51% of the control value after 2, 4 and 10 hr of incubation, respectively. After incubation with 0.1, 0.5, 1 and 2 mM cDDP for 4 hr, the protein SH content represented 86, 68, 58 and 49% of the control values, respectively. The time courses of protein-bound SH groups in BBM vesicles exposed to cDDP yielded similar patterns (fig. 4), whatever the chloride ion concentration in the incubation solution. After exposure to a given concentration of cDDP (0.1, 0.5 or 1 mM) for 2, 4 or 6 hr, the decrease in protein-bound SH groups in BBM vesicles was not statistically different according to the chloride ion concentration.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of cDDP on protein-bound SH groups in BBM vesicles. BBM vesicles were incubated at 37°C without () or with 0.1 (), 0.5 (), 1 () or 2 mM () cDDP in 150 mM NaCl (A), 30 mM NaCl (B) or NaCl-free (C) solution. In control BBM vesicles, the protein SH content was 82.5 ± 1.2, 83.5 ± 0.9 and 83.0 ± 1.2 µmol/g protein in 150 mM NaCl, 30 mM NaCl and NaCl-free solutions, respectively. The data represent the mean ± S.E.M. of three separate preparations of BBM vesicles.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Platinum binding to BBM vesicles. BBM vesicles were incubated at 37°C with 0.1 (), 0.5 (), 1 () or 2 mM () cDDP in 150 mM NaCl (A), 30 mM NaCl (B) or NaCl-free (C) solution. The data represent the mean ± S.E.M. of three separate preparations of BBM vesicles.

Modulation of cDDP-induced effects by DDTC. Na⁺-dependent MGP uptake, protein-bound SH groups and platinum binding were measured in BBM vesicles incubated for 4 hr in the presence or absence of cDDP, followed by a 1-hr postincubation period with 2 mM DDTC in 150 mM KCl or KCl-free solution (figs. 6 and 7). Postincubation of BBM vesicles in the absence of DDTC for 1 hr did not modify Na⁺-dependent MGP uptake, protein SH content or platinum binding relative to values obtained after incubation with 0.75 mM cDDP in 150 mM KCl solution or 0.5 mM cDDP in KCl-free solution. The DDTC concentration used in this study did not change the protein SH content or Na⁺-dependent MGP uptake relative to control BBM vesicles in 150 mM KCl or KCl-free solution. After 4 hr of exposure to 0.75 mM cDDP in 150 mM KCl solution and 0.5 mM cDDP in KCl-free solution, Na⁺-dependent MGP uptake represented 26 and 24% of the control values, respectively (fig. 6A and 7A). A 1-hr postincubation period with 2 mM DDTC after cDDP exposure led to 94% and 138% increases in Na⁺-dependent MGP uptake measured after 4 hr of incubation with 0.75 mM cDDP in 150 mM KCl solution and 0.5 mM cDDP in KCl-free solution, respectively (figs. 6A and 7A); in both cases this corresponded to Na⁺-dependent MGP uptake of approximately 55% compared with the control value. In parallel, a 1-hr postincubation period with 2 mM DDTC led to reductions of 11 and 24 µmol/g protein in platinum binding to BBM vesicles incubated for 4 hr with 0.75 mM cDDP in 150 mM KCl solution and 0.5 mM cDDP in KCl-free solution, respectively (figs. 6C and 7C). DDTC did not significantly modify the cDDP-induced decreases in protein SH content in BBM vesicles incubated in 150 mM KCl or KCl-free solution (figs. 6B and 7B). After 1 hr of postincubation with 2 mM DDTC, the SH_L/Pt_B molar ratio was close to 1 in BBM vesicles incubated both with 0.75 mM cDDP in 150 mM KCl solution and with 0.5 mM cDDP in KCl-free solution.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Modulation of the effects of cDDP by DDTC in the presence of chloride ions. Na+-dependent uptake of 1 mM MGP (A), protein-bound SH groups (B) and platinum binding (C) were measured in BBM vesicles incubated for 4 hr in the absence (control 150 mM KCl) or presence of 0.75 mM cDDP, followed by a 1-hr incubation with 2 mM DDTC in 150 mM KCl solution. The data represent the mean ± S.E.M. of three to four separate preparations of BBM vesicles. a, significantly different from control; b, significantly different from incubation in the absence of DDTC, Student's paired t test.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Modulation of the effects of cDDP by DDTC in the absence of chloride ions. Na+-dependent uptake of 1 mM MGP (A), protein-bound SH groups (B) and platinum binding (C) were measured in BBM vesicles incubated for 4 hr in the absence (control KCl-free) or presence of 0.5 mM cDDP, followed by a 1-hr incubation with 2 mM DDTC in KCl-free solution. The data represent the mean ± S.E.M. of three separate preparations of BBM vesicles. a, significantly different from control; b, significantly different from incubation in the absence of DDTC, Student's paired t test.

DDTC binding to BBM vesicles. After 1 hr of postincubation with 2 mM DDTC of BBM vesicles incubated for 4 hr with 0 to 1 mM cDDP in 150 mM KCl or KCl-free solution, DDTC binding to BBM vesicles was proportional to platinum binding and represented 1.9 times (r = 0.99) the platinum binding to BBM vesicles both in 150 mM KCl and in KCl-free solutions (fig. 8).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   DDTC binding to BBM vesicles. BBM vesicles were incubated at 37°C for 4 hr with cDDP (0.1-1 mM), followed by a 1-hr incubation with 2 mM DDTC in 150 mM KCl () or KCl-free () solution. The data represent the mean ± S.E.M. of three to four separate preparations of BBM vesicles.

	Discussion

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Effects of cDDP on BBM vesicles. cDDP inhibited low-affinity Na⁺-coupled glucose uptake in BBM vesicles from the renal cortex. The cDDP-induced inhibition of Na⁺-coupled glucose uptake is consistent with a direct interaction between cDDP and the transport protein; cDDP did not modify BBM fluidity, which is associated with changes in the Na⁺/glucose system (Courjault-Gautier et al., 1995) and the volume of BBM vesicles (data not shown). In the presence of an extracellular-like concentration of chloride ions (150 mM), the cDDP-induced inhibition of Na⁺-dependent glucose uptake was associated with platinum binding and decrease in protein SH content in BBM vesicles, with an SH_L/Pt_B molar ratio close to 1 at cDDP concentrations below 1 mM. Our results strongly suggest that the cDDP-induced inhibition of Na⁺-dependent uptake of glucose, which possesses SH group(s) that are essential for the activity of the transport protein (Lo and Silverman, 1994), may result from platinum binding to protein-bound SH groups. Mercury chloride, which binds covalently to SH groups, inhibits Na⁺-dependent glucose uptake in BBM vesicles from the renal cortex (Kintziger and Kuntziger, 1986). Our results are consistent with the decrease in protein-bound thiols in the renal cortex and the glucosuria which occur at a very early stage in rats exposed to cDDP (Goldstein et al., 1981; Mistry et al., 1991). Moreover, in rats treated with cDDP, the decline in protein-bound SH content is higher in the subcellular fractions from the renal cortex, which contains the highest platinum concentration (Levi et al., 1980). However, our results suggest that, at high cDDP concentrations (1 mM and above), cDDP can interact with groups other than protein-bound thiols, because the SH_L/Pt_B molar ratio was below 1 in the presence of high chloride ion concentrations. cDDP, which has been shown to react with highly reactive sulfur-containing nucleophiles such as protein-bound thiols by direct displacement of the chloride ligands (Borch et al., 1988; Nagai et al., 1996), could also interact with the sulfur atom of methionine residues when protein-bound thiols are saturated. Several platinum (II) complexes have been shown to bind to the sulfur atom of methionine residues of proteins (Howe-Grant and Lippard, 1980; Melius and Friedman, 1977; Pizzo et al., 1986).

Influence of chloride ion concentration on cDDP-induced effects. The formation of electrophilic hydrated complexes of cDDP is far more abundant in medium containing low chloride ion concentrations (Howe-Grant and Lippard, 1980). The mass spectrometry analysis of cDDP solutions showed that, in our experimental conditions, cDDP remained in its dichloro form in the presence of a high chloride ion concentration (extracellular-like concentration), whereas cDDP hydrated complexes were formed in solution containing a low chloride ion concentration (intracellular-like concentration) and in chloride-free solution. This agrees with the theoretical calculations on the different chemical forms of cDDP obtained from the equilibrium constants reported by Jennerwein and Andrews (1994).

Modulation of cDDP-induced effects by DDTC. DDTC, a sulfur-based chemoprotective agent, has been shown to reduce cDDP-induced nephrotoxicity in several animal studies and in patients with cancer when administered after the anticancer drug (Pinzani et al., 1994; Treskes and van der Vijgh, 1993). Treatment with DDTC immediately after cDDP exposure has also been shown to lift the cDDP-induced inhibition of -glutamyltranspeptidase activity in BBM vesicles from rat renal cortex and to remove platinum bound to specific biological nucleophiles (Bodenner et al., 1986). Our results show that incubation of BBM vesicles with DDTC after exposure to cDDP in the presence or absence of a high chloride ion concentration suppressed approximately 34 and 45% of the cDDP-induced inhibition of Na⁺-coupled glucose uptake (corresponding to approximately 55% of the control value) and reduced platinum binding to BBM vesicles by approximately 29 and 45%, respectively. In these experimental conditions, DDTC did not restore the protein SH content of BBM vesicles exposed to cDDP in the presence or absence of a high chloride ion concentration, as reported previously (Lempers and Reedijk, 1990). The failure of DDTC to restore protein-bound SH groups in cDDP-treated BBM vesicles did not arise from a substitution of bound platinum by DDTC. DDTC, which has been shown to remove platinum bound to some biological nucleophiles, resulting in the formation of Pt(DDTC)₂ (Lempers and Reedijk, 1990), did not bind to control BBM vesicles but directly interacted with the remaining bound platinum, with a DDTC/platinum stochiometry of 2:1, in cDDP-treated BBM vesicles. Moreover, these effects of DDTC were associated with an increase in the SH_L/Pt_B molar ratio which became close to 1 in BBM vesicles incubated with DDTC after exposure to cDDP in the presence or absence of a high chloride ion concentration. These findings strongly suggest that 1) the marked remaining inhibition of Na⁺-coupled glucose uptake by cDDP after partial restoration by DDTC results from a direct interaction of cDDP and/or its hydrated forms with the SH groups essential for the activity of the transport protein and 2) partial restoration by DDTC of cDDP-induced inhibition of Na⁺-coupled glucose uptake results from platinum removal from other nucleophilic groups than the SH group of cysteine residues. The DDTC-induced decreases in platinum binding to BBM vesicles may involve, at least in part, the removal of platinum bound to the sulfur atom of methionine residues, in keeping with the displacement of platinum bound to Pt-methionine protein adducts by DDTC (Lempers and Reedijk, 1990).