Glutathione Conjugates Recognize the Rossmann Fold of Glyceraldehyde-3-phosphate Dehydrogenase*

(Received for publication, December 13, 1996, and in revised form, January 28, 1997)

Mark Puder Dagger and Roy J. Soberman Dagger §

From the Dagger  Arthritis Unit and the Department of Medicine, Massachusetts General Hospital, Massachusetts General Hospital East, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 µM ATP and greater than 50% with 100 µM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4.


INTRODUCTION

Leukotriene (LT)1 C4 and the biologically active prostaglandins (PG) are synthesized at specific intracellular locations and then move to sites where they are exported from cells. 5-Lipoxygenase is targeted to the nuclear membrane (1-3) where, in conjunction with 5-lipoxygenase-activating protein (FLAP), it catalyzes the sequential conversion of arachidonic acid to both (5S)-HETE and to LTA4 (4, 5). LTA4 is then conjugated with either reduced glutathione to form LTC4 by the enzyme LTC4 synthase (6-8), or acted on by the enzyme LTA4 hydrolase to form LTB4 (9). Based on the structural similarity between LTC4 synthase and FLAP, the intracellular location where this reaction most likely takes place is on the inner leaflet of the nuclear membrane (8). LTC4 formed at this location must then traverse the cell until it comes into association with the multidrug resistance-associated protein (10, 11), located in the plasma membrane (12), which mediates its export and that of other glutathione conjugates.

The requirement for intracellular movement and subsequent export is shared with arachidonic acid metabolites of the PG family. PGH synthase-2 is located with its active site facing the interior of the nucleus (13, 14), so that PGH2 formed at this site must be exported from the nucleus before it is acted on by enzymes catalyzing the formation of the bioactive PGs (e.g. PGE2 and PGD2). These products in turn are exported from cells. PGD2 can also interact intracellularly with the gamma  form of the peroxisomal proliferator-activated receptor (15-17) to regulate adipocyte development.

These considerations suggest that LTC4 and glutathione conjugates formed intracellularly can potentially interact with a variety of intracellular proteins. To begin to understand the structural basis of these interactions we employed two photoaffinity ligands, S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) and LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA), to identify the binding folds of proteins of membrane-associated proteins, which could recognize LTC4 and other glutathione conjugates. In studies with inside-out red blood cell vesicles, the major protein binding both probes was found to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Photoaffinity labeling of HL-60 cell cytosol also identified GAPDH as a protein binding LTC4.

Isolation and sequencing of iodinated peptides from the erythrocyte membrane protein and V8 protease digestion of labeled, homogeneous GAPDH indicated that both probes bound to the NAD+ binding pocket, or Rossmann fold (18, 19). The data suggest that GAPDH may also function as a not previously recognized class of proteins which bind LTC4 and glutathione conjugates.


EXPERIMENTAL PROCEDURES

Reagents

ATP, AMP, AMP-PCP, beta -NAD+, S-(p-nitrobenzyl)glutathione, glyceraldehyde-3-phosphate, L-cysteine, Ponceau S, Trizma base, 2-mercaptoethanol, concanavalin A-agarose, Coomassie Brilliant Blue R-250, and GAPDH from human erythrocytes, Dulbecco's minimal Eagle's medium, and fetal bovine serum were purchased from Sigma. MgCl2·6H2O, NaOH, NH4HCO3, methanol, acetic acid, SDS, glycerol, sodium arsenate (Fisher), 6% dextrose 70 in 0.9% sodium chloride (Kendall-McGraw), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) (Pierce), LTC4, (BioMol), V8 protease (sequence grade), ultrapure sucrose, beta -octyl glucoside, protein G-Sepharose (Boehringer Mannheim), lysyl endopeptidase (Wako), dibasic sodium phosphate, and sodium pyrophosphate (Mallinckrodt) were purchased. Mini Protean II Ready gels (Tris-glycine, 10%, and Tris-Tricine, 16.5%), kaleidoscope polypeptide standards, prestained low molecular weight standards, Silver Stain Plus, Tricine, glycine, Bio-Rad protein assay, Tricine loading buffer (Bio-Rad), Solvable tissue solubilizer, Formula 989 scintillation mixture (Packard), Centricon 10 concentrators (Amicon), Immobilon-P polyvinylidene difluoride (PVDF) membranes, HA filters with pore size 0.45 µm (Millipore), reflection NEF film (DuPont NEN) were obtained from the respective suppliers. Antibodies to GAPDH were kindly provided by Dr. Michael Sirover, Temple University. Antibody to Rb(C36) was obtained from Santa Cruz Biotechnology. ECL was purchased from DuPont NEN.

Radiochemicals and Photoaffinity Ligands

8-Azido-[alpha -32P]ATP (350 GBq/mmol) (ICN), [3H]LTC4 (4.9 GBq/mmol) (DuPont NEN) were purchased. PNBG-[125I]ASA and LTC4-[125I]ASA were synthesized by the New England Nuclear custom iodination group. Ten µg of NHS-ASA was iodinated by the IODOGEN method and prepared to a final radioactivity concentration of 16.4 mCi/0.6 ml. The iodinated NHS-ASA was split in two, and one half coupled to 100 µg of S-(p-nitrobenzyl)glutathione for 2.5 h at room temperature and the product isolated by reverse phase-high performance liquid chromatography (RP-HPLC). Alternatively, the second half of the iodinated NHS-ASA was coupled to 50 µg of LTC4 overnight (Nicholson et al., 20) and the product isolated by RP-HPLC.

Preparation of Red Blood Cell Vesicles

Twenty ml of peripheral venous blood was obtained from healthy donors, mixed with 3 ml of acid citrate and 4 ml of 6% dextran 70 in 0.9% NaCl, and allowed to sediment by gravity. The supernatant was discarded and the cells were washed three times with five volumes of ice-cold phosphate-buffered saline and pelleted at 2300 × g for 10 min after each wash. Hemolysis was performed in 40 volumes of 5 mM NaPO4 on ice for 2 min. Erythrocyte ghosts were pelleted at 22,000 × g for 10 min. Two additional wash cycles were performed. Membranes were vesiculated by resuspending the pellet in 0.5 mM NaPO4, pH 8.0, at 4 °C for 12 h. The membranes were centrifuged at 28,000 × g, resuspended in 0.5 mM Tris buffer, pH 7.5, and passed through a 27-gauge needle five times. Sixty mg of concanavalin A-agarose was equilibrated with 0.5 mM Tris and mixed with 20 mg of the membrane protein solution (50%, v/v) in a 50-ml conical tube and gently agitated for 10 min at room temperature (21, 22). The mixture was then poured into a 12 × 1.5-cm column, and the inside-out vesicles were washed through with the same buffer. The vesicles were then collected by centrifugation at 20,000 × g for 20 min. Protein concentrations were determined by the method of Bradford (23).

Culture of HL-60 Cells and Preparation of Subcellular Fractions

HL-60 cells were cultured to a density of 106/ml in Dulbecco's minimal Eagle's medium with 10% fetal bovine serum. Cells were then collected by centrifugation at 1000 × g for 5 min and then washed three times in PBS. They were then resuspended at a concentration of 107/ml in either 0.25 M sucrose, 10 mM Tris HCl, 2 mM EDTA with 5 µg/ml leupeptin, 1 µg/ml apoprotinin, 0.7 µg/ml pepstatin, 1 mM EDTA for Western blotting or in 50 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 µg/ml leupeptin, 1 µg/ml apoprotinin, 0.7 µg/ml pepstatin, 1 mM EDTA for photoaffinity labeling. For Western blotting the cells were disrupted to greater than 90% in a Potter-Elvehjem homogenizer and then centrifuged first at 800 × g to remove unbroken cells and debris. The post-nuclear supernatant was centrifuged at 12,000 × g for 10 min, and the resulting supernatant was centrifuged at 100,000 × g for 1 h. The resulting pellet was resuspended in 1 ml of 250 mM sucrose, 10 mM Tris HCl, pH 7.4, and then repeatedly aspirated through a 27-gauge needle to disperse it. For photoaffinity labeling the cells were disrupted in a sonicating water bath and then centrifuged as above. Microsomes were resuspended in labeling buffer.

Membrane Vesicle Transport-

LTC4 and other glutathione conjugates are transported across membrane vesicles in an ATP-, Mg2+-, and temperature-dependent manner (10, 11, 21, 22). To determine the active transport of photoaffinity ligands, 6 pmol of PNBG-[125I]ASA or 3 pmol of LTC4-[125I]ASA was mixed with 10 mM Tris, pH 7.5, and 250 mM sucrose in the presence or absence of 4 mM ATP and 20 mM MgCl2. The mixture was preincubated on ice or at 37 °C for 1 min. Uptake was initiated by the addition of 400 µg of erythrocyte membranes in a total volume of 250 µl. Fifty-µl aliquots were taken at time zero, 1, 5, 10, and 30 min, diluted in 2 ml of ice-cold buffer, 10 mM Tris, pH 7.5, containing 250 mM sucrose, and immediately passed through presoaked nitrocellulose filters using a rapid filtration apparatus (Millipore). The filters were washed twice with 5 ml of ice-cold buffer. The filter radioactivity was then measured in a gamma  counter. In control experiments ATP was replaced with 4 mM AMP or 4 mM AMP-PCP.

GAPDH Assays

Glyceraldehyde-3-phosphate dehydrogenase was assayed spectrophotometrically by following the reduction of NAD+ at 340 nm (24). The assay contained 30 mM sodium pyrophosphate, 12 µm sodium arsenate, and 4 mM cysteine, pH 8.4. GAPDH (0.4 µg) was incubated in the above reaction mixture for 60 s and then mixed with varying concentrations of NAD+ (10-100 µM) for 30 s. The reaction was initiated by the addition of 150 µM glyceraldehyde-3-phosphate in a final reaction volume of 1 ml at 25 °C pH 8.4. All points were determined in duplicate at 30 s for calculations of initial velocity (V0) using a Beckman DU-64 spectrophotometer by measuring the change in absorbance at 340 nm or with the use of a molar extinction coefficient of 6.3 × 103. For inhibitor studies varying concentrations of SPNBG (100 µM to 1 mM) were included in the reaction mixture before the addition of GAPDH.

Photoaffinity Labeling

Fifty µg of RBC membrane vesicles were mixed with 0.4 pmol of PNBG-[125I]ASA, 1.0 pmol of LTC4-[125I]ASA, or 75 pmol of 8-azido-[alpha -32P]ATP in 0.5 mM Tris buffer, pH 7.5 (final volume of 250 µl), in a 1.5-ml Eppendorf tube with the cap removed, chilled on ice for 10 min, and then exposed to long wave UV light (360 nm) for 7 min. The mixture was centrifuged at 14,000 × g and the supernatant discarded. The membranes were then solubilized in 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.001% bromphenol blue. Proteins were then separated by 10% SDS-PAGE using Bio-Rad Tris-glycine minigels and identified by either staining with 1% Coomassie Brilliant Blue or by silver staining. Photoaffinity-labeled proteins were subsequently identified by autoradiography. Experiments labeling HL-60 cytosol with LTC4-[125I]ASA were performed identically except that 250 µg of protein were used.

Electroblotting and Sequencing

Photoaffinity-labeled erythrocyte membrane proteins were separated by 10% SDS-PAGE and electroblotted to PVDF membranes in 25 mM Tris, 192 mM glycine buffer, pH 8.3, 20% methanol, in a Bio-Rad mini blotting apparatus at 100 V (constant) for 1 h. The membranes were stained with a solution of 0.5% Ponceau S in 1% acetic acid for 5 min and then destained with 1% acetic acid until the background was minimal, and then evaluated by autoradiography. Approximately 16 µg of the photoaffinity-labeled 38-kDa protein was transferred to PVDF membranes. The membrane containing the protein was cut out and the protein digested in situ with lysyl endopeptidase at the Whitehead Protein Sequencing Facility. The peptide fragments were separated by RP-HPLC (HP1090 M) using a Vydac 2.1 × 250-mm C18 column and fractions collected by hand. Radiolabeled peaks were identified by gamma  counting of each fraction and were analyzed on a gas phase microsequencer (model 475, Applied Biosystems, Inc.) at the Whitehead Protein Sequencing Facility. Peptide searches were made using the BLAST program.

HL-60 proteins were separated by SDS-PAGE and blotted as above, except that nitrocellulose membranes were used. For Western blotting these were then blocked in 3% nonfat dried milk in PBS for 1 h at room temperature and then washed for 10 min three times in PBS. Antibody was added in 3% nonfat dried milk in PBS and allowed to react for 1 h at room temp. Following washing, the membranes were developed by ECL and exposed for 1 min prior to development.

Immunoprecipitation

250 µg of dialyzed cytosol was photoaffinity-labeled as described above. After labeling, the cytosol was concentrated to 30 µl in a Centricon concentrator and then suspended to a volume of 1 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 µg/ml leupeptin, 1 µg/ml apoprotinin, 0.7 µg/ml pepstatin, 1 mM/ml EDTA. Immunoprecipitation was then performed using anti-GAPDH antibody and anti-Rb(C36) as a control following the protein G-agarose protocol of Boehringer Mannheim. Immunoprecipitated proteins were analyzed by SDS-PAGE followed by autoradiography.

Competition

Five µg of purified erythrocyte GAPDH was chilled on ice with 2.0 pmol of PNBG-[125I]ASA or 5 pmol of LTC4-[125I]ASA, and varying concentrations of unlabeled competitors in a volume of 100 µl of 0.5 mM Tris, pH 7.5. The mixture was photolyzed and analyzed by 10% SDS-PAGE, Coomassie Blue staining, and autoradiography.

V8 Protease Digestion

Four µg of erythrocyte GAPDH was labeled with either PNBG-[125I]ASA or LTC4-[125I]ASA as described above. The buffer was exchanged to a final volume of 100 µl of 50 mM NH4HCO3 and 15 mM KCl by three cycles of dilution to 2 ml and concentration to 100 µl in a Centricon 10 concentrator. The protein was digested with 4 µg of V8 protease for 2 h at 37 °C. Digested or undigested GAPDH was mixed with sample buffer containing 200 mM Tris, pH 6.8, 40% glycerol, 2% SDS, 2% 2-mercaptoethanol, and 0.04% Coomassie Brilliant Blue G-250. The samples were heated for 5 min at 95 °C and the peptides and/or undigested enzyme resolved in 16.5% Tris/Tricine minigels (18). Protein and peptides were identified by silver staining or by staining with Coomassie Brilliant Blue. Photoaffinity-labeled peptides and proteins were then identified by autoradiography.

[3H]LTC4 Labeling, Gel Elution, and Counting

[3H]LTC4 (8.5 KBq, 20 nM) was incubated with 4 µg of erythrocyte GAPDH in 0.5 mM Tris, pH 7.5, for 10 min at 37 °C, frozen in liquid nitrogen, and irradiated at 360 nm for 7 min (11). The labeled protein was mixed with SDS loading buffer, heated for 5 min at 95 °C, analyzed by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue. To locate the peptide incorporating the radioactivity, stained bands were excised and incubated with 0.5 ml of deionized water and 0.5 ml of Solvable for 3 h at 50 °C, mixed with Formula 989 scintillation mixture, and then radioactivity quantitated by scintillation counting in a Packard 1500 Tri-Carb liquid scintillation counter.


RESULTS

To determine the glutathione conjugate binding sites of proteins that regulate trafficking of LTC4 and other glutathione conjugates, we identified membrane proteins that were photoaffinity-labeled with both of the probes shown in Fig. 1, and also with 8-azido-[alpha -32P]ATP. The latter was chosen to identify the nucleotide binding site of potential ATP-binding cassette carrier proteins. The membrane initially chosen for these studies and to test the probes was human erythrocyte inside out vesicles. Erythrocyte membranes export glutathione conjugates (21, 22), contain the multidrug resistance-associated protein (25), are simple in composition, and are available in large quantities. We initially determined whether the photoaffinity ligands were actively transported by erythrocyte membranes. The uptake of PNBG-[125I]ASA and LTC4-[125I]ASA both increased over a period of 1-5 min in the presence of ATP and then leveled off over the next 25 min (data not shown). In the absence of ATP, or Mg2+, or when AMP-PCP or AMP was substituted for ATP no uptake was observed. When 3 pmol of PNBG-[125I]ASA was used as a substrate, 157 fmol (mean, n = 2) was taken up at 1 min, whereas in the absence of ATP only 43.5 fmol was associated with the vesicles. When 6 pmol of LTC4-[125I]ASA was added in a reaction, 194 fmol was taken up at 1 min, whereas 36 fmol was associated with vesicles in the absence of ATP. These data indicated that these ligands were actively imported by erythrocyte membrane vesicles.


Fig. 1. Structure of iodinated photoaffinity ligands. A, S-(p-nitrobenzyl)glutathione-[125I]ASA. B, leukotriene C4-[125I]ASA.
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Erythrocyte membranes were then incubated on ice with PNBG-[125I]ASA or LTC4-[125I]ASA, photolyzed with UV light, separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and analyzed by autoradiography (Fig. 2A). One major band of 38 kDa was observed to be photoaffinity-labeled with LTC4-[125I]ASA (lane 1) or PNBG-[125I]ASA (lane 2). A minor band was observed at 190 kDa under both circumstances, and at 80 kDa using LTC4-[125I]ASA as the probe. Labeling with 8-azido-[alpha -32P]ATP showed multiple bands (Fig. 2B, lane 1), but included one at 38 kDa. Lane 2 shows that a single band is identified in these membranes by labeling with PNBG-[125I]ASA.


Fig. 2. Photoaffinity labeling of erythrocyte membrane vesicles. A, 50 µg of erythrocyte vesicles and either 1.0 pmol of LTC4-[125I]ASA (lane 1) or 0.4 pmol of PNBG-[125I]ASA (lane 2) were mixed in 250 µl of 0.5 mM Tris, pH 7.5, and chilled for 10 min at 0 °C. The mixture was then irradiated at 360 nm for 7 min. After centrifugation at 14,000 × g, the pellet was dissolved in SDS loading buffer and analyzed by 10% SDS-PAGE and autoradiography. B, 50 µg of erythrocyte vesicles was photoaffinity-labeled with either 75 pmol of 8-azido-[alpha -32P]ATP (lane 1) or 0.4 pmol of PNBG-[125I]ASA (lane 2) and the results analyzed as described above.
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To identify the prominent 38-kDa protein, erythrocyte membranes were labeled with PNBG-[125I]ASA and then electroblotted to PVDF membranes. After staining with Ponceau S, ~16 µg of protein was digested in situ with lysyl endopeptidase and the resulting peptides separated by RP-HPLC. Fractions corresponding to OD peaks were collected by hand and then analyzed by gamma  counting. Two fractions (54 and 56) were found to contain radioactivity. Sequencing was able to identify the 11 N-terminal amino acids of the peptide collected in peak 54. When analyzed by the BLAST program, these amino acids could be aligned with amino acids 119-129 of human liver GAPDH as shown in Table I, with an identity of 7/11 residues. When the peptide in Peak 56 was analyzed in the same manner, it could be aligned with an identity of 11/11 amino acids for amino acids 67 to 77 of liver GAPDH (Table I). Both peptides are contained in the NAD+ binding region of the protein (18, 19).

Table I.

Comparison of the amino acid sequences of iodinated peptides with GAPDHs


A.
GAPDH (liver)  V   I   I   S   A   P   S   A   D   A   P 
 |    |    |          |    |               |     |
Peak 54  V   I   I   T   A   P   E   D   D   A   L
             |          |    |               |     |
GAPDH (muscle)  I   V   I   S   A   P   S   A   D   A   P
Amino acid no. 119 120 121 122 123 124 125 126 127 128 129
B.
GAPDH (liver)  L   V   I   N   G   N   P   I   T   I   F
 |    |    |   |    |   |    |   |    |     |    |
Peak 56  L   V   I   N   G   N   P   I   T   I   F
 |    |   |         |             |   |     |    |
GAPDH (muscle)  L   V   I   D   G   K   A   I   T   I   F
Amino acid no.  67  68  69  70  71  72  73  74  75  76  77

To further confirm that the nucleotide binding site was photoaffinity-labeled, 5 µg of purified GAPDH were photoaffinity-labeled with PNBG-[125I]ASA in the presence of increasing concentrations of NAD+ or ATP (Fig. 3). PNBG-[125I]ASA photolabeling was inhibited ~50% by 10 µM ATP and completely at a concentration of 100 µM. One hundred µM NAD+ inhibited labeling by this compound by greater than 50%, and 10 mM NAD+ almost completely inhibited labeling. Photoaffinity labeling of GAPDH by LTC4-[125I]ASA was inhibited by ~50% with 1 µM ATP and almost completely at 10 µM ATP. Photoaffinity labeling with this probe was essentially abolished at 10 µM and 100 µM NAD+. To confirm that glutathione conjugates that were not derivatized as photoaffinity labels interacted with the active site of GAPDH, we determined the Ki for S-(p-nitrobenzyl)glutathione of GAPDH enzymatic activity. Fig. 4 shows the results of one of two experiments. For two experiments the Ki was found to be 379 µM ± 6 (mean ± S.D., n = 2).


Fig. 3. Inhibition of photoaffinity labeling of GAPDH. Five µg of human erythrocyte GAPDH was chilled at 0 °C for 10 min with the indicated concentrations of NAD+ and ATP prior to photoaffinity labeling. The reaction was then mixed with SDS loading buffer and analyzed by SDS-PAGE and autoradiography.
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Fig. 4. Inhibition of GAPDH activity. Secondary plot of slopes versus S-(p-nitrobenzoyl)glutathione concentration used to determine the Ki. The slopes were obtained from from Lineweaver-Burke plots calculated at different concentrations of S-(p-nitrobenzoyl)glutathione.
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Because the peptide sequence of the 38-kDa protein was obtained with membranes labeled with the PNBG-[125I]ASA probe, we utilized the ability of V8 protease to cleave GAPDH into its NAD+ and glyceraldehyde-3-phosphate binding domains to provide additional confirmation that LTC4-[125I]ASA bound to the Rossmann fold. Four micrograms of enzyme were photoaffinity-labeled with either PNBG-[125I]ASA or LTC4-[125I]ASA. Two micrograms of each labeled aliquot were digested with V8 protease. Digested and undigested enzyme were resolved by 16.5% Tris/Tricine gels and then analyzed by silver staining (Fig. 5A) followed by autoradiography (Fig. 5B). Undigested enzyme labeled with LTC4-[125I]ASA or PNBG-[125I]ASA is represented by a single band of 38 kDa when analyzed by either silver staining or autoradiography. When digestion with V8 protease was performed for 2 h, two major fragments could be identified by silver staining at ~8.8 and 6.8 kDa, the latter known to contain the NAD+ binding site (18). GAPDH was cleaved by V8 protease so that radioactivity from both probes was identically distributed. A minority was localized to the remaining uncut protein, and the majority associated with the 6.8-kDa fragment containing the NAD+ binding site. The ability of [3H]LTC4 to label GAPDH was then examined by the same approach except that bands were labeled with Coomassie Blue and then protein extracted with Solvable prior to analysis by beta -scintillation counting. In two experiments, all the radioactivity was distributed identically to that of the photoaffinity ligands, indicating that a natural ligand bound to the same site as the photoaffinity label (data not shown).


Fig. 5. PNBG-[125I]ASA and LTC4-[125I]ASA bind to the Rossmann fold of GAPDH. Four µg of GAPDH was photoaffinity-labeled with either PNBG-[125I]ASA or LTC4-[125I]ASA and then digested with 4 µg of V8 protease for 2 h at 37 °C. Digested (lanes 1 and 3) and undigested (lanes 2 and 4) GAPDH were separated by 16.5% Tris/Tricine-PAGE, and analyzed by either silver staining (A) and autoradiography (B).
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We next determined whether GAPDH in nucleated cells could interact with LTC4-[125I]ASA. In initial experiments, HL-60 cell fractions were analyzed by Western blotting to determine the distribution of GAPDH. Small amounts were found in the membrane fraction, and greater than 96% in the cytosol. Cytosol was then dialyzed against labeling buffer, labeled with LTC4-[125I]ASA, and then analyzed by SDS-PAGE and autoradiography. Two bands were identified, one with the identical size to GAPDH at 38 kDa and a second at~ 27 kDa (Fig. 6). To confirm the identity of the 38-kDa protein, labeled cytosol was precipitated with antibody to GAPDH. A single band was found at 38 kDa (Fig. 6C), which was not seen with control antibody. Although GAPDH was found in the membrane fraction, this could not be successfully labeled. In addition, this small fraction could only be dissociated by the use of detergent, unlike the erythrocyte enzyme, which is released by salt (26).


Fig. 6. Photoaffinity labeling and identification of GAPDH in HL-60 cytosol. Two µg of GAPDH (lane 1) or 250 µg of HL-60 cytosol (lanes 2-4) were photoaffinity-labeled with LTC4-[125I]ASA and then analyzed by SDS-PAGE with autoradiography. Lanes 1 and 2 were analyzed directly and lanes 3 and 4 after immunoprecipitation with either anti-GAPDH (lane 3) or anti-Rb(C36) (lane 4).
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DISCUSSION

Photoaffinity labeling of inner erythrocyte membranes by PNBG-[125I]ASA or LTC4-[125I]ASA (Fig. 1) showed one predominant protein at 38 kDa and a second less intense band at 190 kDa (Fig. 2A). When analyzed by one-dimensional electrophoresis, the predominant 38-kDa protein appeared to also be labeled with 8-azido-[32P]ATP (Fig. 2B). The strategy of labeling with two different probes was employed to identify members of the ATP-binding cassette family. However, carrier proteins that move midsize molecules contain at least six transmembrane domains and are at least 60 kDa in size (27). The size of 38 kDa was considered too small for such a protein. Because of its prominence, the 38-kDa band photolabeled with PNBG-[125I]ASA was sequenced directly, with the assumption that even if there were several co-migrating proteins, labeled peptides could be isolated after digestion and sequenced. This approach proved viable in that only two peptides were identified after lysyl endopeptidase digestion and isolation by RP-HPLC. Peak 56 had 100% identity to liver GAPDH, whereas peak 54 was identical with 7/11 amino acids. As shown in Table I, the degree of identity between liver and muscle GAPDH is similar (8/11) to that between liver GAPDH and peak 54, further suggesting that the labeled protein is erythrocyte GAPDH (which has yet to be cloned).

The identification of the binding of LTC4-[125I]ASA as well as PNBG-[125I]ASA to the 6.8-kDa V8 protease fragment further indicates that both labels bind to the same site (Fig. 5). The relevance to the natural ligands is demonstrated by the fact that [3H]LTC4 photolabels the same 6.8-kDa peptide as the iodinated probes. Three lines of evidence indicate that both photoaffinity labels bind to the NAD+ binding site of GAPDH. First, the peptide mapping is the same; second, the binding of both probes is competitively inhibited with both NAD+ and ATP (Fig. 3); third, the parent compounds of the probes; and fourth, S-(p-nitrobenzyl)glutathione competitively inhibits GAPDH activity. When peptide mapping was performed, LTC4-125I-ASA and PNBG-[125I]ASA were localized to the fragment that contains the 62 N-terminal amino acids (Fig. 5). The identification of this peptide has been previously confirmed by sequencing (18). However, the two peptides identified by direct sequencing in this report lie C-terminal to this region, but still within the NAD+ binding site. One possible explanation for this is that either the membrane associations or protein interactions of GAPDH determine the orientation of PNBG-[125I]ASA and LTC4-[125I]ASA within the Rossmann fold, allowing them to cross-link to different residues. The ability of GAPDH to alter the properties of its catalytic site depending on its environment is supported by the observations that the independent 38-kDa subunits function as a DNA glycosylase rather than a glycolytic enzyme (28) and that the ability of the enzyme to bind RNA is determined by its redox state (18). Alternatively, GAPDH may have a membrane association with a specific integral membrane protein, which could bind and then pass the labels to the Rossmann fold. GAPDH has been shown to associate with erythrocyte Band 3 (29) and the glucose transporter (30); however, no protein corresponding to the size of either carrier was labeled. Therefore, if such a mechanism were operative, then the transfer to GAPDH would have be both highly efficient and specific in terms of the orientation of the proteins.

The ability of GAPDH to serve as a binding protein for LTC4 in nucleated cells was demonstrated in HL-60 cytosol (Fig. 6). Although the identity of the second labeled protein of 27 kDa was not pursued, it is possible that it is the human homologue of the rat Ya glutathione S-transferase subunit which has been shown to bind LTC4 (31). The lack of labeling of the small membrane-bound component of GAPDH could represent a further change in enzyme configuration of this tightly bound form so that binding cannot occur.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants ES-50859 (to R. J. S.) and CA-09580 and CA-09031 (to M. P.).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 correspondence should be addressed: Rm. 8306, Massachusetts General Hospital East, 149-The Navy Yard-13th St., Charlestown, MA 02129. Tel.: 617-726-3747; Fax: 617-726-5651; E-mail: soberman{at}helix.mgh.harvard.edu.
1   The abbreviations used are: LT, leukotriene; (5S)-HETE; (5S)-Hydroxy-6-cis-8,11,14-trans-eicosatetraenoic acid; FLAP, 5-lipoxygenase-activating protein; PNBG-[125I]ASA, S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid; LTC4-[125I]ASA, LTC4-[125I]4-azidosalicylic acid; PG, prostaglandin; AMP-PCP, beta ,gamma -methyleneadenosine 5'-triphosphate; NHS-ASA, N-hydroxysuccinimidyl-4-azidosalicylic acid; PVDF, polyvinylidene difluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RP-HPLC, reverse phase-high performance liquid chromatography; PBS, phosphate-buffered saline; Tricine, N-tris (hydroxymethyl)methylglycine.

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