Localization of the Iodomycin Binding Site in Hamster P-glycoprotein*

(Received for publication, April 17, 1997, and in revised form, May 30, 1997)

Annette Demmer Dagger §, Hubert Thole Dagger , Peter Kubesch Dagger , Tanja Brandt Dagger , Manfred Raida par , Rainer Fislage Dagger and Burkhard Tümmler Dagger

From the Dagger  Klinische Forschergruppe, Zentrum Biochemie and Zentrum Kinderheilkunde, OE 4350, Medizinische Hochschule Hannover, D-30623 Hannover,  Max-Planck-Institut für experimentelle Endokrinologie, D-30625 Hannover and par  Niedersächsisches Institut für Peptidforschung, D-30625 Hannover, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. Plasma membrane vesicles isolated from multidrug-resistant Chinese hamster ovary B30 cells were photolabeled with [125I]iodomycin. After chemical cleavage behind the tryptophan residues, 125I-labeled peptides were separated by electrophoresis and high performance liquid chromatography. Edman sequencing revealed that [125I]iodomycin had been predominantly incorporated into the fragment 230-312 of isoform I of hamster P-glycoprotein. According to models based on hydropathy plots, the amino acid sequence 230-312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the N-terminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A.


INTRODUCTION

P-glycoprotein, which belongs to the large family of ABC1 transporters (1), binds and transports a broad range of structurally unrelated compounds (2). Its overexpression may cause the phenomenon of multidrug resistance (MDR) during cancer chemotherapy, whereby the tumor cells become resistant to a variety of antineoplastic agents due to a reduced intracellular accumulation of drugs (2-4). The MDR phenotype can be overcome by modulators, i.e. substances that are bound by P-glycoprotein and inhibit its drug excluding function (3). The medically important substrates of P-glycoprotein comprise anticancer drugs such as Vinca alkaloids and anthracyclines (2-4) and approved drugs that turned out to be potent modulators such as verapamil (5, 6), calcium antagonists (7), and cyclosporins (8).

The major issue of how P-glycoprotein can handle so many substrates has been mainly approached by photoaffinity labeling and mutagenesis studies. Mutants that arose spontaneously during drug selection of cells and thereby changed their resistance profile were detected in the first cytoplasmic loop of human MDR1 protein (Gly185 right-arrow Val) (9) and in transmembrane segment (TM) 6 of hamster P-glycoprotein (Gly338 right-arrow Ala/Ala339 right-arrow Pro) (10). Active mutagenesis identified further motifs and positions in the N- and C-terminal half of P-glycoprotein which influence substrate specificity (11, 12). For example, the exchange of amino acids in the first cytoplasmic loop (11), TM5, TM6, TM10, TM12 (13-16), and the cytosolic linker peptide (17) resulted in all in an altered multidrug resistance phenotype, suggesting that during binding and transport the substrate is recognized by multiple residues located either in the cytosolic, membraneous, or ectoplasmic domains.

Hydropathy plots deduced from the primary sequence predict that P-glycoprotein consists principally of two symmetrical halves, each of which contain a membrane domain with six membrane-spanning segments and a subsequent cytosolic nucleotide binding fold (18-20). The topology of P-glycoprotein in vivo, however, may be more variable than predicted, e.g. under certain experimental conditions, TM segments were detected outside the membrane and loops postulated to face the cytosol were found on the extracellular side (21-25).

Although the three-dimensional structure of the drug binding pocket(s) is unknown, substantial progress has been made in the localization of the binding site(s) in the primary sequence by mutagenesis (see above) and by photoaffinity labeling. Photoreactive derivatives of cyclosporin (26), daunomycin (27), verapamil (28), prazosin (29), Vinca alkaloids (30, 31), and calcium channel blockers (32) have been shown to become photoincorporated into P-glycoprotein. Typically the sensitivity and specificity of the interaction between drugs and P-glycoprotein has been substantiated by the inhibition of photoaffinity labeling by substrates of P-glycoprotein such as vinblastine or verapamil. The affinity of cytotoxic agents to compete for the photobinding site decreases for most photoreactive compounds, in the order vinblastine > daunomycin > colchicine (29). In the case of the photoreactive 1,4-dihydropyridine derivatives azidopine (29, 33, 34), iodoarylazidoprazosin (35-37), and AIPP-forskolin (38), the binding sites were allocated by immunoreactivity of enzymatic digests to epitope-specific antibodies in or close to TM6 and TM12, i.e. domains of the molecule which, from structure-function comparisons with other ABC transporters, are assumed to be directly involved in the transport of substrate (12).

Iodomycin is the Bolton-Hunter derivative of the inherently photoreactive anthracycline daunomycin (27) (Fig. 1). Iodomycin binds to P-glycoprotein in plasma membrane vesicles and intact cells with an affinity of 107 to 108 M-1 (39) and belongs to the compounds to which multidrug-resistant cells are cross-resistant, i.e. the accumulation of iodomycin is reduced in P-glycoprotein-overexpressing cells compared with their drug-sensitive parents (27). The anthracycline recombines with its binding sites in a diffusion-controlled reaction with kon rates of (2-4) × 109 M-1 s-1. P-glycoprotein is specifically photolabeled by [125I]iodomycin (27). Since the photoreaction is several orders of magnitude slower than the formation of the non-covalent drug-protein complex, the thermodynamic affinity constant determines the concentration-dependent yield of photoincorporation of iodomycin into protein and the competitive reduction of photobinding of radioactive [125I]iodomycin by increasing concentrations of non-radioactive anthracycline (39). Vinblastine and cyclosporin compete strongly with iodomycin for its photobinding site in P-glycoprotein, whereas colchicine and nifedipine are poor inhibitors of the photobinding (27). Photolabeling of protein is optimal at 488 nm, which coincides with the absorption maximum of anthracene derivatives (39), implying that the photoreaction of iodomycin with amino acids is different from that of azido compounds (28-32).


Fig. 1. The structure of [125I]iodomycin. Daunomycin reacts with 125I-Bolton-Hunter reagent to [125I]iodomycin.
[View Larger Version of this Image (16K GIF file)]

In this study we describe the localization of the major binding site for [125I]iodomycin in multidrug-resistant CHO B30 cells. Photolabeled P-glycoprotein was cleaved behind its tryptophan residues with BNPS-skatol (40), and the resulting peptides were purified by electrophoresis and HPLC. Edman sequencing revealed that the binding site for iodomycin resides in a region in the N-terminal half of the protein that is distinct from the binding site described for [125I]iodoarylazidoprazosin (37).


MATERIALS AND METHODS

Cell Culture

CHO B30 cells were grown in alpha -minimum essential medium supplemented with glutamine, nucleosides, 10% calf serum, and 30 µg/ml colchicine (41).

Preparation of Plasma Membrane Vesicles

For the isolation of plasma membranes, culture medium was replaced by 1 mM EGTA, 2 mM EDTA, 5 mM dithiothreitol, 20 mM Hepes buffer, pH 7.4. Cells from 50 tissue culture plates (diameter, 9 cm) were scraped off with a rubber policeman and disrupted by ultrasonication until 80% of the cells were lysed as checked by light microscopy. After removal of cell debris by centrifugation (4,000 × g for 10 min), the membraneous fraction of the supernatant was collected by centrifugation at 40,000 × g for 1 h. The pellet was subsequently centrifuged for 90 min (40,000 × g) through a 35% sucrose cushion. After resuspension of the interphase in 5 mM Tris buffer, pH 7.5, the plasma membrane fraction was collected by a spin of 100,000 × g for 1 h. The protein pellet was solubilized in 5 mM Tris-HCl, pH 7.5, with 8.6% sucrose (w/v) and stored at -70 °C. The yield varied between 1 and 2 mg of plasma membrane protein. During the whole procedure, the following antiproteases were added to avoid any protein degradation: leupeptin (final concentration 4 µM) (Sigma), N-p-tosyl-L-arginine-methyl ester (final concentration 26 µM) (Sigma), and Pefabloc (final concentration 0.4 mM) (Merck).

The Purification of P-glycoprotein with an Agarose Gel System

Metaphor agarose (FMC) was dissolved in 500 mM Tris, 160 mM borate, pH 8.5, to give a concentration of 7% for the resolving gel (2/3) and 3.5% for the stacking gel (1/3). The boiled agarose was casted vertically in a 12.5 × 8.5 × 0.2-cm large chamber consisting of two prewarmed scratched glass plates. A maximum of 450 µg of ethanol-precipitated membrane protein dissolved in 62.5 mM Tris-HCl pH 6.8; 7.5% (w/v) FicollTM type 400; 0.001% (w/v) bromphenol blue, and 7 mg/ml dithiothreitol was loaded onto the agarose gel. Electrophoresis was run at 8 V/cm for 1 h and then 12 V/cm for 3 h at 4 °C. The anodal buffer consisted of 90 mM Tris, pH 8.5, 90 mM borate, and 0.1% SDS. The cathodal buffer consisted of 9 mM Tris, pH 8.5, 9 mM borate, and 0.1% (w/v) SDS. The agarose stripe containing P-glycoprotein was cut out of the gel and chopped into small pieces, collected in an Eppendorf tube, covered with a 10-fold excess of buffer (50 mM Tris, pH 10.4, 3 mM EDTA, 0.5% (w/v) N-lauroylsarcosinate), and melted at 80 °C for 15-30 min until the suspension appeared homogeneous. The samples were solidified in an ice bath and stored for 3 h at -70 °C. The agarose was thawed on ice and centrifuged for 30 min at 4 °C at 13,000 × g. The agarose was pelleted, whereas P-glycoprotein remained dissolved in the supernatant (freeze-squeeze procedure). To remove any residual agarose and detergent and to increase protein concentration, the solution was centrifuged through a 100,000-Da cut-off filter in a Centricon tube (Amicon).

Western Immunoblot

The presence of P-glycoprotein was detected on immunoblots of 6% PAGE-separated membrane proteins (42) with polyclonal rabbit antiserum mdr (AB-1) (Oncogene Science) or monoclonal antibody C219 (Signet) as the primary antibody and CPDSTAR chemiluminescence in strict accordance with a protocol of the manufacturer (Tropix). Purified P-glycoprotein was also detected on immunoblots with the primary mouse monoclonal antibody C494 (Signet).

Photoaffinity Labeling with [125I]Iodomycin

[125I]Iodomycin (2000 Ci/mmol) was prepared from daunomycin by reaction with 125I-labeled Bolton-Hunter reagent (ICN) and subsequently purified as described previously (27) (Fig. 1). The absence of contamination with unreacted Bolton-Hunter reagent and daunomycin in the purified product was controlled by thin layer chromatography (27). For photoaffinity labeling, maximal 75 µg of plasma membrane protein from CHO cells and 125I-labeled iodomycin (0.075 pmol for the competition experiment and 0.75 pmol for the cleavage experiments) were suspended in 150 µl in a quartz cuvette and were illuminated for 15 min with visible light, emitted from a 500-watt xenon lamp that had passed through two 3-cm filters of water and a saturated aqueous CuSO4 solution. For the competition experiment with cyclosporin A, variable concentrations of the drug were added prior to the photoreaction. Protein was precipitated with ice-cold ethanol (final 70% (v/v)) at -20 °C for 4 h and analyzed by gel electrophoresis (42) or stored at -70 °C until further processing.


Fig. 2. Photoaffinity labeling of P-glycoprotein in plasma membranes of CHO B30 cells. 50 µg of plasma membrane protein were labeled with 0.05 pmol of [125I]iodomycin in the presence of various concentrations of cyclosporin A (lanes 1-5 (from left to right): 0, 3 nM, 30 nM, 300 nM, and 3 µM). The binding of iodomycin was documented by 1-week exposure of the dried 6% PAGE gel to a Kodak X-Omat film. 300 nM cyclosporin A or more displaces iodomycin out of its binding site.
[View Larger Version of this Image (67K GIF file)]

Detection of Radioactivity

For the detection of radioactivity the gels were either exposed to Kodak X-Omat AR film or to a Fuji imaging plate type BAS-IIIs. Liquid samples of [125I]iodomycin-labeled proteins and peptides were measured in a Compu gamma  counter.

Cleavage of Protein

Mild tryptic fragmentation (43) cleaves P-glycoprotein in plasma membranes into two large fragments. Plasma membrane protein was digested with trypsin in a volume of 100 µl and an enzyme to a substrate ratio of 1:50 for 30 min at 37 °C.

Chemical Fragmentation

Plasma membrane proteins were cleaved either with N-chlorsuccinimide (44) or BNPS-skatol (40). For the cleavage procedure with N-chlorsuccinimide, 75 µg of plasma membrane protein in 150 µl were incubated immediately after the photoreaction for 30 min at 37 °C with 75 µl of H2O and 150 µl of the following mixture: 1 mg of N-chlorsuccinimide dissolved in 500 µl of H2O, 500 mg of urea, and 500 µl of acetic acid. The reaction products were precipitated with ice-cold ethanol and analyzed on a 4%/12% discontinuous Tricine-PAGE (45). For the cleavage procedure with BNPS-skatol, the precipitated protein was dissolved in 7.5 µl of H2O and 22.5 µl of the following mixture: 1.3 mg of BNPS-skatol (Fluka) dissolved in 1 ml of 100% acetic acid. The reaction was carried out at 47 °C for 18 h. The digest was stopped by diluting the mixture with 24 µl of H2O. The cleavage products were concentrated in an evaporator and analyzed on a 4%/12% discontinuous Tricine-PAGE. For a preparative gel, the 15 cm × 17 cm × 0.1-cm gel was loaded with 2 mg of total membrane protein or 200 µg of purified P-glycoprotein.

Purification of the [125I]Iodomycin-labeled Skatol Fragment

According to the signals generated by photoimaging, the 125I-labeled peptide of lowest molecular weight was gel-eluted onto a 3-kDa cut-off membrane in the Centrilutor (Amicon, catalog no. 1047601) for 12 h at 80 V and 4 °C in a glycine buffer (42). Next, excess volume was removed by centrifugation of the Centricon tubes at 4 °C at 7,500 × g. This solution was washed three times by ultrafiltration with the maximum possible volume of 5 mM Tris-HCl, pH 7.5, to remove SDS to the greatest possible extent. Subsequently the concentrated peptide solution was removed from the filtration unit and supplemented with guanidinium hydrochloride to a final concentration of 6 M. The membranes of the Centricon units were washed each with 15 µl of 6 M guanidinium hydrochloride in 5 mM Tris-HCl to avoid any loss of peptide. The collected fractions were separated on an Aquapore phenyl 7-µm 30 × 2.1-mm Brownlee column by HPLC reversed phase chromatography (Applied Biosystems 0711-0068). The linear discontinuous gradient was generated by mixtures of solution A (0.1% trifluoroacetic acid) and B (60% acetonitrile, 20% 2-propanol; 0.085% trifluoroacetic acid) in Milli Q water: 1 min 1-10% B; 5 min 10-25% B; 50 min 25-50% B, 65 min 50-65% B; 70 min 65-70% B; 85 min 70-99% B (Applied Biosystems/BAI HPLC system, flow rate 0.1 ml/min, detection 214 nm).

Edman Sequence Analysis

Sequence analysis was carried out with the Applied Biosystems model 477A apparatus according to Bökenkamp et al. (46).


RESULTS

The Cell Line CHO B30

This P-glycoprotein-overexpressing cell line (41) grown in medium supplemented with 30 µg/ml colchicine has a P-glycoprotein content of 10-20% of total membrane protein (Fig. 3A), which makes B30 cells a suitable source for the large scale purification and characterization of P-glycoprotein.


Fig. 3. Purification of P-glycoprotein with an agarose gel system. A, lanes 1-3, 6% PAGE of 50 µg of plasma membrane protein of CHO B30 cells (lane 3) preincubated with endoglycosidase F (lanes 1 and 2). Note the shift of the P-glycoprotein band in the Coomassie stain by deglycosylation. Lane 4, silver stain of 1 µg of P-glycoprotein purified with the agarose gel system. B, immunoblot of purified P-glycoprotein. Lanes 1 and 5, 2.5 µg; lanes 2 and 6, 5 µg; lanes 3 and 7, 3 µg of purified protein. C, 6 µg of plasma membrane protein of CHO B30 cells. Lanes 1-3, immunoreactivity of P-glycoprotein with the polyclonal antibody mdr AB-1; lanes 5-7, immunoreactivity of P-glycoprotein with the monoclonal antibody C494, which only recognizes human and hamster P-glycoproteins of class I. Occasionally an extra 110-kDa band was detected by the polyclonal antibody (left panel), but not by C494 (right panel).
[View Larger Version of this Image (75K GIF file)]

Purification of P-glycoprotein

To facilitate the identification of cleavage products of digested P-glycoprotein (see below), we developed a simple and fast protocol to purify this very hydrophobic membrane protein. Electrophoretic separation of membrane proteins in Metaphor® agarose (47) and recovery of P-glycoprotein by a freeze-squeeze procedure in the presence of the detergent N-lauroylsarcosine turned out to be a powerful tool to isolate P-glycoprotein at a preparative scale. 450 µg of plasma membrane protein yielded between 20 and 40 µg of 70-99% pure P-glycoprotein, whereby both yield and purity depended on strict adherence to the protocol (Fig. 3A, lane 4).

Fig. 3B shows the immunoblot of purified iodomycin-labeled P-glycoprotein with polyclonal antibody mdr AB-1 (Fig. 3B, left) and monoclonal antibody C494 (Fig. 3B, right). The latter antibody only recognizes human and hamster P-glycoproteins of class I (48).

Photoaffinity Labeling with Iodomycin

Iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin (Fig. 1), belongs to the compounds to which multidrug-resistant CHO cells are cross-resistant (27). The inherently photoreactive [125I]iodomycin is incorporated with high specificity into P-glycoprotein in plasma membranes of CHO B30 cells (Fig. 2, lane 1). Photolabeling was competitively inhibited by typical substrates of P-glycoprotein with differential efficacy; for example, more than 10 µM colchicine, but less than 0.5 µM vinblastine or cyclosporin (Fig. 2) were necessary to cause 50% reduction of [125I]iodomycin photobinding (17, 27, 39, 49). According to these data, the photobinding site of P-glycoprotein for iodomycin represents a high affinity site for cyclosporin and vinblastine, but a low affinity site for colchicine.

Mild Tryptic Digestion

Our next step was to investigate in which part of the protein the photoreaction takes place. According to the protocol published by Georges et al. (43), it is possible to cleave P-glycoprotein in plasma membrane vesicles by mild tryptic digestion into a ~110-kDa N-terminal fragment and a ~65-kDa C-terminal fragment. Both fragments were detected in immunoblots by monoclonal antibody C219, which recognizes a common epitope in the two nucleotide binding folds (data not shown). [125I]Iodomycin predominantly labeled the 110-kDa N-terminal fragment and only a minor portion of radioactive drug was photoincorporated into the C-terminal fragment (Fig. 4).


Fig. 4. Mild tryptic digest. Autoradiogram of a dried 6% PAGE gel is shown. Iodomycin-labeled P-glycoprotein in plasma membrane vesicles was tryptically cleaved into two large fragments according to the protocol by Georges et al. (43). Lane 1 (50 µg) and lane 3 (25 µg), iodomycin-labeled plasma membrane protein (not digested with trypsin); lane 2, 50 µg of plasma membrane protein digested with trypsin. Iodomycin is predominantly photoincorporated into the ~100-kDa N-terminal fragment (2-week exposure to a Kodak X-Omat film).
[View Larger Version of this Image (46K GIF file)]

The Chemical Cleavage of the Iodomycin-labeled P-glycoprotein

After the major photobinding site had been assigned to the N-terminal half of P-glycoprotein, its localization was refined by internal cleavage at the tryptophan residues with N-chlorsuccinimide (NCS) or BNPS-skatol. Since the amino acid sequence of hamster P-glycoprotein contains only 11 tryptophan residues (Table I), the chemical cleavage of Trp-X peptide bonds was expected to result in fewer fragments than the enzymatic digestion with one of the commonly employed endoproteinases.

Table I. The tryptophan cleavage pattern of P-glycoprotein (pgp1)

The table represents the total digestion fragments of P-glycoprotein with tryptophan cleavage reagents and their localization in the primary sequence of hamster P-glycoprotein (pgp1).

No. N-terminal amino acid C-terminal amino acid Molecular weight

1 1 44 5343
2 45 133 9635
3 134 159 3093
4 160 209 5566
5 210 229 2062
6 230 312 9045
7 313 695 42,214
8 696 705 1244
9 706 800 10,824
10 801 852 5366
11 853 1105 27,797
12 1106 1276 18,812

Treatment of [125I]iodomycin-photolabeled plasma membranes (Fig. 5, A and B) or purified P-glycoprotein (Fig. 5C) with NCS (Fig. 5A) or BNPS-skatol (Fig. 5, B and C) yielded a pattern of photolabeled peptides that was more complex than predicted by the primary sequence of P-glycoprotein for a total digest at the tryptophan residues. This finding could result from (a) nonspecific cleavage, (b) partial digestion, (c) more than one photobinding site in P-glycoprotein, and/or (d) labeling of further proteins. Explanation (a) is unlikely to apply because despite their distinct mechanisms of cleavage both NCS and BNPS-skatol gave the same pattern of radiolabeled peptides (cf. Fig. 5, A and B). Partial digestion of protein, however, had to be seriously considered because the reaction was found never to proceed to completion. To differentiate between partial digestion and/or multiple photobinding sites, BNPS-skatol digests of total plasma membrane protein (Fig. 5B) and purified P-glycoprotein (Fig. 5C) were compared in their profile of photolabeled fragments. Two bands equivalent to fragments of about 50-60 kDa and smaller than 10 kDa were seen in autoradiograms from both the plasma membrane preparation and purified P-glycoprotein (cf. Fig. 5, B and C), indicating that they represent iodomycin-labeled peptides of P-glycoprotein. The <10-kDa radiolabeled fragment was observed in all cleavage experiments. The hamster pgp1 cDNA sequence (10) predicts that such a small skatol digestion fragment exists only in the N-terminal half of P-glycoprotein (Table I), which is in accordance with the outcome of the mild tryptic digest whereby most [125I]iodomycin was photoincorporated into the N-terminal fragment (see above). When the cleavage with BNPS-skatol was extended from 4 h to 18 h, the signal of the 50-60-kDa peptide decreased and that of the small <10-kDa peptide increased (Fig. 5B). The mutual dependence of the yield of the two radiolabeled P-glycoprotein subfragments on reaction time indicates that the 50-60-kDa peptide is a partial digestion fragment which contains the <10-kDa peptide as the internal [125I]iodomycin-labeled complete digestion fragment. This interpretation is consistent with the molecular weights of complete and partial digestion fragments predicted by the primary amino acid sequence of hamster P-glycoprotein (fragment 6 versus fragment [5]-6-7, Table I). The nature of the other radiolabeled fragments (Fig. 5, A and B) could also be interpreted as partial digestion fragments from the N-terminal half of pgp, whereby fragment 6 was an obligatory constituent. Since partial digestion dissipated the 125I-labeled photoproducts on multiple peptides, any minor photobinding site, for example that assigned by mild tryptic digestion to the C-terminal half of P-glycoprotein (see above), was not detectable by radioimaging after cleavage with NCS or BNPS-skatol.


Fig. 5. Chemical cleavage of P-glycoprotein behind tryptophan residues resolved on a 12% Tricine-PAGE. A, iodomycin-labeled plasma membrane vesicles were cleaved with N-chlorsuccinimide. Lanes 1-3, 75, 150, and 300 µg of plasma membrane protein. The smallest iodomycin-labeled peptide is a fragment with a mass between 8 and 10 kDa. Since [125I]iodomycin is photoincorporated to the greatest extent into P-glycoprotein and not in any other CHO plasma membrane protein (cf. Fig. 3), the strong signals of 125I-labeled fragments in the 8-56-kDa molecular mass range generated by NCS cleavage are most likely partial digestion fragments of P-glycoprotein. B, iodomycin-labeled plasma membrane vesicles were cleaved with BNPS-skatol. Lanes 5 and 6, 250 µg of plasma membrane proteins were digested for 4 h (lane 5) and 18 h (lane 6) with BNPS-skatol. The 4-h digest in acetic acid without the cleavage reagent BNPS-skatol (lane 4) served as the control. The smallest iodomycin-labeled peptide was again a fragment with a mass of between 8 and 10 kDa. Note the increase of the smallest labeled peptide at the expense of all other bands by prolongation of the reaction time. In particular, the intensity of the 56-kDa band decreased. BNPS-skatol yields less partial digestion fragments than NCS. C, 40 µg of iodomycin-labeled purified P-glycoprotein was digested with BNPS-skatol for 18 h. Cleavage of purified P-glycoprotein reproducibly yielded only the 8-10-kDa and 56-kDa peptides, whereas all other fragments obtained by incubation of crude plasma membranes (cf. A and B) were not detectable. See Table I for the possible origin of complete and partial digestion fragments of 8-10 or 56 kDa.
[View Larger Version of this Image (64K GIF file)]

The Purification of the Smallest Labeled Peptide

Finally we wanted to identify the fragment in the N-terminal half of the protein where the photoreaction takes place. [125I]Iodomycin-photolabeled substrate was digested with BNPS-skatol, because the cleavage with NCS produced N-terminal blocked fragments not suitable for Edman sequence analysis. The smallest labeled peptide was purified by the combination of gel electrophoresis, electroelution and reversed phase chromatography as described under "Materials and Methods." The chromatography step was necessary to remove all peptides that comigrated with the labeled peptide in the one-dimensional gel. The photocross-linked peptide was eluted in fractions containing more than 60% solvent B. Fig. 6 shows the elution profiles for solvent, absorbance, and radioactivity (marker for [125I]iodomycin). To differentiate between free and covalently bound iodomycin, all 125I-positive fractions were tested by gel electrophoresis and subsequent radioimaging. Radioactivity was associated with the peptide that eluted within the fraction of 60% solvent B. The sample with highest radioactivity was subjected to Laemmli gel electrophoresis, blotted onto a PVDF membrane, stained with Coomassie Blue, and finally subjected to imaging plate analysis (Fig. 7).


Fig. 6. HPLC reversed phase chromatography. Two mg of plasma membrane protein were labeled with [125I]iodomycin, digested with BNPS-skatol for 18 h, and separated by 12% Tricine-PAGE. After exposure of the gel to the imaging plate (24 h), the smallest labeled peptide was cut out of the gel, electroeluted, and separated by reversed phase HPLC chromatography. The elution profile was monitored at 214 nm. Beginning with 60% solvent B, the labeled peptide was eluted from the column. The activity of iodomycin-bound peptide was measured in a gamma  counter. The graph with the spread axis demonstrates the activity of the HPLC fractions containing 50% solvent B or more. The two fractions with the highest radioactivity at 60 and 61% B were immediately subjected to Edman sequence analysis. The N-terminal amino acid sequence of the peptide in these fractions is identical with the first 17 amino acids of fragment 6 (amino acids 230-312) in hamster pgp1 (Table I).
[View Larger Version of this Image (40K GIF file)]


Fig. 7. PVDF blot of the HPLC fraction which eluted at 60-61% solvent B and was subjected to Edman sequencing. The HPLC fraction eluting at 60% solvent B was blotted onto a PVDF membrane, stained with Coomassie Blue, and sequenced. A shows the Coomassie-stained band that migrates between 8 and 10 kDa, and B shows its radioactivity analyzed by exposing the PVDF membrane to an imaging plate. Sometimes the peptide smeared, as can be seen in this case.
[View Larger Version of this Image (91K GIF file)]

Edman Sequence Analysis

The HPLC fraction with the highest radioactivity was given either immediately to Edman sequence analysis or first blotted on the PVDF membrane (v.s.) and then sequenced. The Edman reaction was carried out on purified peptides from four independent membrane preparations with identical outcome. Only one peptide was identified in the reaction cycles independently, whether it was sequenced directly from the HPLC fraction or after the transfer to the PVDF membrane. The reaction enabled the reading of the first 17 amino acids of the 125I-photolabeled peptide. The identified amino acid sequence starts in P-glycoprotein (pgp1) behind the tryptophan residue number 229, i.e. 125I-photolabel was incorporated into fragment 6 of pgp1 (amino acids 230-312, Mr 9045, Table I). According to the topological model established from hydrophobicity plots (19) this amino acid sequence starts in the fourth transmembrane domain, encompasses the second cytoplasmic loop, and stops in the fifth transmembrane domain of P-glycoprotein (Fig. 8).


Fig. 8. Topological model of P-glycoprotein. The figure shows the structure of P-glycoprotein predicted by hydrophobicity plots (18-20). Regions that are involved in the binding of [3H]azidopine, [125I]iodoaryl azidoprazosin, and 125I-6-AIPP-forskolin are marked. The binding site for [125I]iodomycin is localized in an amino acid sequence that stretches from TM 4 to TM 5, encompassing the second cytoplasmatic loop.
[View Larger Version of this Image (24K GIF file)]


DISCUSSION

This study reports on the localization of the binding site for iodomycin in hamster P-glycoprotein. Chemical cleavage of iodomycin-photolabeled P-glycoprotein behind the tryptophan residues revealed that the drug had been predominantly incorporated into the pgp1 fragment 230-312. According to the topology predicted by hydropathy plots (18-20) this amino acid sequence 230-312 forms the distal part of TM4, the second cytoplasmic loop, and the proximal part of TM5 in the N-terminal half of P-glycoprotein (Fig. 8).

To identify a drug photobinding site in P-glycoprotein, the tools and methodology had to be carefully selected and optimized, i.e. the requirements were a photoreactive substrate for P-glycoprotein of high specific activity, a cellular source of high P-glycoprotein contents, a site-specific reagent that reliably cleaves membrane proteins without blockage of the N terminus, and a purification procedure that separates hydrophobic peptides and has been minimized for loss of photoproduct and contamination by compounds that could affect protein sequencing. Our current protocol is considered to fulfill these criteria. Iodomycin photolabels P-glycoprotein in cells and plasma membrane vesicles with high sensitivity and specificity. The overexpression of P-glycoprotein in CHO B30 cells was instrumental to photolabeling P-glycoprotein in sufficient amounts so that 125I-photoproducts were detectable by radioimaging throughout the whole purification procedure. Enzymatic and chemical means were tested for cleavage of purified P-glycoprotein and crude membrane vesicles. Enzymatic proteolysis was found to be disadvantageous because the self-digestion of enzyme contaminated the sample and impeded purification and sequencing of target peptide. In contrast, site-specific cleavage behind tryptophan with BNPS-skatol enabled low background Edman sequencing of the purified 125I-labeled skatol fragment. Its N-terminal sequence enabled us to discriminate between hamster pgp isoforms and to identify pgp1 as the photobinding acceptor of iodomycin. In summary, peptide identification by Edman analysis was chosen because the method yields unequivocal data on the localization of the binding site in the primary sequence.

Apart from iodomycin, some other photoreactive compounds have already been used for the photoaffinity labeling of drug binding sites in P-glycoprotein. Azidopine and the photoreactive derivatives of prazosin and forskolin label both halves of P-glycoprotein (34, 36, 38). [3H]Azidopine, [125I]iodoarylazidoprazosin, and probably also 125I-AIPP-forskolin bind to a common domain in P-glycoprotein (29). Bruggemann et al. (34) recovered 60% of azidopine binding from the CNBr fragment (amino acids 198-440) in the N-terminal half of human MDR1. Site-specific antibodies recognized the photobinding sites for prazosin and forskolin (35, 36, 38) in or close to TM 6 and TM 12; unfortunately, however, epitope specificity of some antibodies was dependent on the detergent that was used for the solubilization of enzymatic digests of photolabeled protein (36). The role of the C-terminal half of P-glycoprotein could be more clearly discerned in studies on MDR1 (class I)/MDR3 (class III) chimera (37). TM 12 and the loop connecting TM 11 and TM 12 were found to contain determinants for substrate specificity and photobinding of [125I]iodoarylazidoprazosin. The distinct localization of photobinding sites for prazosin and iodomycin may reflect different reaction pathways of photolabeling (50) and/or the presence of more than one binding site for the interaction of P-glycoprotein with its substrate.

Tamai and Safa (51) showed that the interaction of vinblastine with the azidopine binding site was noncompetitive. Cyclosporin A competitively inhibited Vinca alkaloid binding, but inhibited azidopine binding in a noncompetitive manner (52). These data suggested the existence of two binding sites on P-glycoprotein, one specific for Vinca alkaloids, verapamil, and cyclosporins and one binding site specific for azidopine. Ayesh et al. (53, 54) extended the model whereby they proposed two different binding sites. One is recognized by substances such as vinblastine, mefloquine, and tamoxifen and the other one by substances such as verapamil. Cyclosporin A may interact with both sites (53). An even more complex view arose from photolabeling studies with a novel 1,4-dihydropyridine derivative (55). Multiple chemosensitizer domains were identified in P-glycoprotein, which were distinct from the Vinca alkaloid binding site. In contrast to these data, Bruggemann et al. (34) found that vinblastine and azidopine bind to a single site in human MDR1. Vinblastine was only able to prevent azidopine binding if a valine was at position 185 instead of a glycine, which occurred during selection of cells with colchicine (9). These data indicate the direct contact of photobinding sites with substrate specificity influencing amino acids. Friche et al. (56) examined the effect of anthracycline analogues on photolabeling of P-glycoprotein by iodomycin and azidopine. Their data suggest that anthracyclines and Vinca alkaloids may bind to a common site in P-glycoprotein and that azidopine and iodomycin label P-glycoprotein with different binding affinities and/or at different sites. The latter view is supported by binding studies with prazosin. This drug inhibited photoaffinity labeling of P-glycoprotein by azidopine (29), but did not displace iodomycin out of its binding pocket (29, 36).2 The major photobinding site of arylazidoprazosin was localized in and C-terminal to TM6 and TM12 and a second minor site between TM4 and up to TM6 (36), of which the latter corresponds with the iodomycin binding site described in this report (Fig. 8).

Although photoaffinity labeling studies provided strong evidence that P-glycoprotein has more than one binding site for drugs and modulators, it is not definite proof because of the inherent limitations of the methodology. Photoreactions with arylazido groups under UV irradiation and iodomycin with visible light share no common pathways, and correspondingly other reaction centers in the protein are selected, which may be part of the same binding site or another one (50, 57). On the other hand, this differential photoreactivity has the advantage of localizing various epitopes of the three-dimensional binding pocket(s), albeit the number of sites remains elusive. The latter issue is more appropriately addressed by analysis of substrate interaction at equilibrium (39, 58).

Since covalent photolabels just indicate the binding domain by primary sequence, supplementary information about the topology of the protein and its functional states is instrumental for the characterization of the binding site(s). According to the topological model of P-glycoprotein predicted from the hydropathy plot (18-20), the binding site for iodomycin is localized in a sequence that starts at amino acid 230 in TM 4, comprises the second cytoplasmatic loop, and stops at amino acid 312 in TM 5 (Fig. 8).

Recent experimental work (59-61) applying up-to-date methodology to full-length P-glycoprotein confirms the structure of P-glycoprotein established by hydropathy plot analysis. Topologies of P-glycoprotein different than predicted have been observed in cell-free (21, 22, 62), Xenopus oocyte (22), and bacteria (23, 24) expression systems. Despite the fact that all these topologies were found in truncated molecules or fusion proteins, Zhang et al. (25) found a structure of full-length P-glycoprotein different from that predicted by hydropathy plot analysis in CHO B30 cells, which express P-glycoprotein naturally by selection with 30 µg/ml colchicine. They probed the structure of P-glycoprotein with site-specific antibodies and found TM 5 and the loop connecting TM 4 and 5 located extracellularly.

The inconsistent topology may be not only a consequence of different experimental set-up, but could also reflect a dynamic structure of P-glycoprotein associated with different functional states. A paradigm is the colicin Ia channel (63). A mobile segment crosses the membrane from one side to the other as the channel opens. These data support the hypothesis that reversible posttranslational changes of the topology may be an essential mechanism of a channel or transporter like P-glycoprotein.

The loop connecting TM 4 and TM 5 contains a sequence motif that is highly conserved among ABC transporters. The EAA or EAA-like motif (EXAXXXG) predicts substrate specificity of prokaryotic ABC transporters (64-66). Substitution of the central glycine causes transporter dysfunction in bacteria (67) and the exchange of the conserved Glu for Asp and Gly for Pro resulted in a complete loss-of-function of PXA1, a gene product of Saccharomyces cerevisiae homologous to the adrenoleukodystrophy protein, an ABC transporter in the membrane of human peroxisomes (66, 68, 69). The conserved motif is also found in P-glycoproteins and is localized in the second cytoplasmatic loop (Fig. 8) connecting TM 4 and 5 at positions Glu279, Ala281, and Gly285 of hamster pgp1 (10). This region resides within the iodomycin-labeled skatol fragment.

In conclusion, this study demonstrates that the binding site for iodomycin is localized in a domain of P-glycoprotein that recognizes vinblastine and cyclosporin with high affinity. The data about the topology of and the conserved sequence motif within the iodomycin-labeled domain suggest a crucial role for this region in the transport function of ABC transporters in general. With respect to P-glycoprotein, the localization of binding site(s) in the primary sequence supplemented with mutagenesis studies and functional studies will identify the constituents of the binding pocket(s) and hence provide a clue as to how P-glycoprotein can recognize and handle such a broad range of structurally unrelated substrates.


FOOTNOTES

*   This work was supported by a grant (to B. T.) from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom all correspondence should be addressed: Klinische Forschergruppe, Zentrum Biochemie, OE 4350, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. Tel.: 49-511-5322920; Fax: 49-511-5325966.
1   The abbreviations used are: ABC, ATP-binding cassette; AIPP-forskolin, 6-O-[[2[3-(4-azido-3-iodophenyl)propionamido]ethyl]carbamyl]forskolin; BNPS-skatol, 2-(2'-nitrophenylsulfenyl)-3-methyl-3'bromoindolenine; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; mdr, multidrug resistance; NCS, N-chlorsuccinimide; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; pgp, hamster P-glycoprotein; PVDF, polyvinylidene difluoride; RT, reverse transcription; TM, transmembrane segment.
2   A. Demmer and T. Brandt, unpublished data.

ACKNOWLEDGEMENTS

We thank our colleagues at the Institut für Peptidforschung (Dr. Forssmann) and at the Max-Planck-Institut für experimentelle Endokrinologie (Dr. Jungblut) for access to their technical facilities, which was fundamental for the success of the project. We are grateful to Manfred Raida's co-workers for their help and Dr. Weißer for his kind advice.


REFERENCES

  1. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113 [CrossRef]
  2. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137-171 [CrossRef][Medline] [Order article via Infotrieve]
  3. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  4. Gottesman, M. M., Pastan, I., and Ambudkar, S. V. (1996) Curr. Opin. Genet. Dev. 6, 610-617 [CrossRef][Medline] [Order article via Infotrieve]
  5. Cano-Gauci, D. F., and Riordan, J. R. (1987) Biochem. Pharmacol. 34, 2115-2123
  6. Dalton, W. S., Grogan, T. M., Meltzer, P. S., Scheper, R. J., Durie, B. G. M., Taylor, C. W., Miller, T. P., and Salmon, S. E. (1989) J. Clin. Oncol. 7, 415-424 [Abstract]
  7. Hofmann, J., Gekeler, V., Ise, W., Noller, A., Mitterdorfer, J., Hofer, S., Utz, I., Gotwald, M., Boer, R., Glossmann, H., and Grunicke, H. H. (1995) Biochem. Pharmacol. 49, 603-609 [CrossRef][Medline] [Order article via Infotrieve]
  8. Sonneveld, P., Durie, B. G. M., Lokhorst, H. M., Marie, J.-P., Solbu, G., Suciu, S., Zittoun, R., Löwenberg, B., and Nooter, K. (1992) Lancet 340, 255-259 [Medline] [Order article via Infotrieve]
  9. Choi, K., Chen, C.-J., Krigler, M., and Roninson, I. B. (1988) Cell 53, 519-529 [Medline] [Order article via Infotrieve]
  10. Devine, S. E., Ling, V., and Melera, P. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4564-4568 [Abstract]
  11. Currier, S. J., Kane, S. E., Willingham, M. C., Cardarelli, C. O., Pastan, I., and Gottesman, M. M. (1992) J. Biol. Chem. 267, 25153-25159 [Abstract/Free Full Text]
  12. Gros, P., Dhir, R., Croop, J., and Talbot, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7289-7293 [Abstract]
  13. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 3143-3149 [Abstract/Free Full Text]
  14. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 19965-19972 [Abstract/Free Full Text]
  15. Loo, T. W., and Clarke, D. M. (1994) J. Biol. Chem. 269, 7243-7248 [Abstract/Free Full Text]
  16. Loo, T. W., and Clarke, D. M. (1994) Biochemistry 33, 14049-14057 [Medline] [Order article via Infotrieve]
  17. Hoof, T., Demmer, A., Hadam, M. R., Riordan, J. R., and Tümmler, B. (1994) J. Biol. Chem. 269, 20575-20583 [Abstract/Free Full Text]
  18. Gros, P., Ben Neriah, Y., Croop, J. M., and Housman, D. E. (1986) Nature 323, 728-731 [Medline] [Order article via Infotrieve]
  19. Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592 [Abstract/Free Full Text]
  20. Chen, C.-J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Cell 47, 381-389 [Medline] [Order article via Infotrieve]
  21. Zhang, J.-T., Duthie, M., and Ling, V. (1993) J. Biol. Chem. 268, 15101-15110 [Abstract/Free Full Text]
  22. Skach, W. R., Calayag, M. C., and Lingappa, V. R. (1993) J. Biol. Chem. 268, 6903-6908 [Abstract/Free Full Text]
  23. Bibi, E., and Béjà, O. (1994) J. Biol. Chem. 269, 19910-19915 [Abstract/Free Full Text]
  24. Béjà, O., and Bibi, E. (1995) J. Biol. Chem. 270, 12351-12354 [Abstract/Free Full Text]
  25. Zhang, M., Wang, G., Shapiro, A., and Zhang, J.-T. (1996) Biochemistry 35, 9728-9736 [CrossRef][Medline] [Order article via Infotrieve]
  26. Foxwell, B. M. J., Mackie, A., Ling, V., and Ryffel, B. (1989) Mol. Pharmacol. 36, 543-546 [Abstract]
  27. Busche, R., Tümmler, B., Riordan, J. R., and Cano-Gauci, D. F. (1989) Mol. Pharmacol. 35, 414-421 [Abstract]
  28. Safa, A. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7187-7191 [Abstract]
  29. Greenberger, L. M., Yang, C.-P. H., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401 [Abstract/Free Full Text]
  30. Safa, A. R., Glover, C. J., Meyers, M. B., Biedler, J. L., and Felsted, R. L. (1986) J. Biol. Chem. 261, 6137-6140 [Abstract/Free Full Text]
  31. Cornwell, M. M., Safa, A. R., Felsted, R. L., Gottesman, M. M., and Pastan, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3847-3850 [Abstract]
  32. Safa, A. R., Glover, C. J., Sewell, J. L., Meyers, M. B., Biedler, J. L., and Felsted, R. L. (1987) J. Biol. Chem. 262, 7884-7888 [Abstract/Free Full Text]
  33. Bruggemann, E. P., Germann, U. A., Gottesman, M. M., and Pastan, I. (1989) J. Biol. Chem. 264, 15483-15488 [Abstract/Free Full Text]
  34. Bruggemann, E. P., Currier, S. J., Gottesman, M. M., and Pastan, I. (1992) J. Biol. Chem. 267, 21020-21026 [Abstract/Free Full Text]
  35. Greenberger, L. M., Lisanti, C. J., Silva, J. T., and Horwitz, S. B. (1991) J. Biol. Chem. 266, 20744-20751 [Abstract/Free Full Text]
  36. Greenberger, L. M. (1993) J. Biol. Chem. 268, 11417-11425 [Abstract/Free Full Text]
  37. Zhang, X., Collins, K. I., and Greenberger, L. M. (1995) J. Biol. Chem. 270, 5441-5448 [Abstract/Free Full Text]
  38. Morris, D. I., Greenberger, L. M., Bruggemann, E. P., Cardarelli, C., Gottesman, M. M., Pastan, I., and Seamon, K. B. (1994) Mol. Pharmacol. 46, 329-337 [Abstract]
  39. Busche, R., Tümmler, B., Cano-Gauci, D. F., and Riordan, J. R. (1989) Eur. J. Biochem. 183, 189-197 [Abstract]
  40. Crimmins, D. L., Mc Court, D. W., Thoma, R. S., Scott, M. G., Macke, K., and Schwartz, B. D. (1990) Anal. Biochem. 187, 27-38 [Medline] [Order article via Infotrieve]
  41. Kartner, N., Evernden-Porelle, D., Bradley, G., and Ling, V. (1985) Nature 316, 820-823 [Medline] [Order article via Infotrieve]
  42. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  43. Georges, E., Zhang, J.-T., and Ling, V. (1991) J. Cell. Physiol. 148, 479-484 [Medline] [Order article via Infotrieve]
  44. Shechter, Y., Patchornik, A., and Burstein, Y. (1976) Biochemistry 15, 5071-5075 [Medline] [Order article via Infotrieve]
  45. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  46. Bökenkamp, D., Jungblut, P. W., and Thole, H. H. (1994) Mol. Cell. Endocrinol. 104, 163-172 [Medline] [Order article via Infotrieve]
  47. Morgan, J. H., Curtis, F. P., and Nochumson, S. (1991) BioTechniques 11, 256-261 [Medline] [Order article via Infotrieve]
  48. Georges, E., Bradley, G., Gariepy, J., and Ling, V. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 152-156 [Abstract]
  49. Demmer, A., Dunn, T., Hoof, T., Kubesch, P., and Tümmler, B. (1996) Eur. J. Pharmacol. 315, 339-343 [CrossRef][Medline] [Order article via Infotrieve]
  50. Glossmann, H., Ferry, D. R., Striessnig, J., Goll, A., and Moosburger, K. (1987) Trends Pharmacol. Sci. 8, 95-100 [CrossRef]
  51. Tamai, I., and Safa, A. R. (1991) J. Biol. Chem. 266, 16796-16800 [Abstract/Free Full Text]
  52. Tamai, I, and Safa, A. R. (1990) J. Biol. Chem. 265, 16509-16513 [Abstract/Free Full Text]
  53. Ayesh, S., Shao, Y.-M., and Stein, W. D. (1996) Biochim. Biophys. Acta 1316, 8-18 [Medline] [Order article via Infotrieve]
  54. Shao, Y.-M., Ayesh, S., and Stein, W. D. (1997) Biochim. Biophys. Acta 1360, 30-38 [Medline] [Order article via Infotrieve]
  55. Boer, R., Dichtl, M., Borchers, C., Ulrich, W. R., Marecek, J. F., Prestwich, G. D., Glossmann, H., and Striessnig, J. (1996) Biochemistry 35, 1387-1396 [CrossRef][Medline] [Order article via Infotrieve]
  56. Friche, E., Demant, E. J. F., Sehested, M., and Nissen, N. I. (1993) Cancer Res. 67, 226-231
  57. Safa, A. R. (1993) Cancer Invest. 11, 46-56 [Medline] [Order article via Infotrieve]
  58. Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, pp. 849-886, W. H. Freeman, San Francisco
  59. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 843-848 [Abstract/Free Full Text]
  60. Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411 [Medline] [Order article via Infotrieve]
  61. Kast, C., Canfield, V., Levenson, R., and Gros, P. (1996) J. Biol. Chem. 271, 9240-9248 [Abstract/Free Full Text]
  62. Zhang, J.-T., and Ling, V. (1991) J. Biol. Chem. 266, 18224-18232 [Abstract/Free Full Text]
  63. Slatin, S. L., Qiu, X.-Q., Jakes, K. S., and Finkelstein, A. (1994) Nature 371, 158-161 [CrossRef][Medline] [Order article via Infotrieve]
  64. Saurin, W., Koster, W., and Dassa, E. (1994) Mol. Microbiol. 12, 993-1004 [Medline] [Order article via Infotrieve]
  65. Shani, N., Watkins, P. A., and Valle, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6012-6016 [Abstract/Free Full Text]
  66. Shani, N., Sapag, A., and Valle, D. (1996) J. Biol. Chem. 271, 8725-8730 [Abstract/Free Full Text]
  67. Koster, W., and Bohm, B. (1992) Mol. Gen. Genet. 232, 399-407 [Medline] [Order article via Infotrieve]
  68. Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990) J. Biol. Chem. 265, 4534-4540 [Abstract/Free Full Text]
  69. Mosser, J., Lutz, Y., Stoeckel, M., Sarde, C., Kretz, C., Douar, A., Lopez, J., Auburg, P., and Mandel, J. (1994) Hum. Mol. Genet. 3, 265-271 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.